Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M708844200 on December 17, 2007

J. Biol. Chem., Vol. 283, Issue 8, 5034-5045, February 22, 2008
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
283/8/5034    most recent
M708844200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Birts, C. N.
Right arrow Articles by Wilton, D. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Birts, C. N.
Right arrow Articles by Wilton, D. C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

A Catalytically Independent Physiological Function for Human Acute Phase Protein Group IIA Phospholipase A2

CELLULAR UPTAKE FACILITATES CELL DEBRIS REMOVAL*Formula

Charles N. Birts, C. Howard Barton, and David C. Wilton1

From the School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, SO16 7PX, United Kingdom

Received for publication, October 26, 2007 , and in revised form, December 17, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human group IIA phospholipase A2 (IIA PLA2) is an acute phase protein first identified at high concentrations in synovial fluid from patients with rheumatoid arthritis. Its physiological role has since been debated; the enzyme has a very high affinity for anionic phospholipid interfaces but expresses almost zero activity with zwitterionic phospholipid substrates, because of a lack of interfacial binding. We have prepared the cysteine-containing mutant (S74C) to allow the covalent attachment of fluorescent reporter groups. We show that fluorescently labeled IIA was taken up by phorbol 12-myristate 13-acetate-activated THP-1 cells in an energy-dependent process involving cell surface heparan sulfate proteoglycans. Uptake concurrently involved significant cell swelling, characteristic of macropinocytosis and the fluorescent enzyme localized to the nucleus. The endocytic process did not necessitate enzyme catalysis, ruling out membrane phospholipid hydrolysis as an essential requirement. The enzyme produced supramolecular aggregates with anionic phospholipid vesicles as a result of bridging between particles, a property that is unique to this globally cationic IIA PLA2. Uptake of such aggregates labeled with fluorescent anionic phospholipid was dramatically enhanced by the IIA protein, and uptake involved binding to heparan sulfate proteoglycans on activated THP-1 cells. A physiological role for this protein is proposed that involves the removal of anionic extracellular cell debris, including anionic microparticles generated as a result of trauma, infection, and the inflammatory response, and under such conditions serum levels of IIA PLA2 can increase ~1000-fold. A similar pathway may be significant in the uptake into cells of anionic vector DNA involving cationic lipid transfection protocols.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The human group IIA PLA22 was first discovered at high concentrations in the synovial fluid of patients with rheumatoid arthritis (1) and has subsequently been shown to be associated with a variety of inflammatory diseases. Serum levels of the secreted enzyme can rise 1000-fold during severe sepsis, and the enzyme has been recognized as an acute phase protein under the transcriptional control of pro-inflammatory cytokine signaling (2). The antibacterial properties of this highly cationic extracellular protein is part of the innate immune response and can be readily explained by its ability to both bind and hydrolyze the anionic cell membranes of bacteria (3-6).

However, despite extensive literature on this subject (7-10), the precise roles of the enzyme remain enigmatic, particularly in terms of effects of this protein on host cell function. This is because in contrast to the very high affinity of this extracellular enzyme for anionic phospholipid interfaces, the enzyme has a very low affinity for the plasma membrane of normal host cells because of the zwitterionic nature of their outer phospholipid monolayers. As a result negligible rates of membrane hydrolysis are observed, although immunoactivated cells and cells that have initiated apoptosis are hydrolyzed (11), and a role for this enzyme in the clearance of apoptotic and damaged cells has been proposed (12).

There is no detailed understanding of how interactions between the extracellular enzyme and the cell surface might affect cell function as part of the inflammatory response. The binding of extracellular human IIA to cell surface HSPGs has been noted but not investigated in detail (8, 13). In addition, human high affinity cell surface receptors for the human group IIA enzyme have not been detected (14, 15). Therefore, it is of considerable interest to investigate the interaction of labeled human IIA with human cells to clarify the role of this clinically important enzyme.

As the group IIA protein targets cells from an extracellular location, chemical labeling of the protein with fluorescent reporters and investigation of its interaction with cells in culture is feasible. The human monocyte-derived cell line THP-1 was chosen as a target, as these cells can be differentiated into a cell with an adherent, macrophage-like phenotype and by a number of procedures, including PMA treatment.

Our data demonstrate the ability of these activated cells to take up the IIA protein rapidly, by a mechanism that does not require enzyme catalysis, and subsequent trafficking of IIA to the cell nucleus was observed. Uptake was HSPG- and energy-dependent, and the highly significant cell swelling observed was characteristic of macropinocytosis. Most importantly the globally cationic protein aggregated anionic phospholipid vesicles and dramatically promoted their uptake into cells. We propose that a fundamental physiological role of the group IIA PLA2 is in the clearance of anionic pathological cell debris, including microparticles resulting from the inflammatory response.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Specialized Reagents—DOPG, DOPC, and DOPS were from Avanti%20Polar%20Lipids">Avanti Polar Lipids, Inc. (Alabaster, AL). 11-(Dansylamino)-undecanoic acid, Alexa Fluor-568 maleimide dye, annexin V-Alexa Fluor-488 conjugate, concanavalin A-Alexa Fluor-488 conjugate, and Texas Red 1,2-dihexadecanoyl-sn-glycero-3 phosphoethanolamine, calcein were from Molecular Probes. Texas Red-2-sulfonamidoethyl-methanethiosulfonate, 2-[(5-fluorosceinyl)aminocarbonyl]ethyl-methanethiosulfonate, and dansylamidoethyl-methanethiosulfonate were from Toronto Research Chemicals (Toronto, Canada).

Cell Culture—THP-1 cell line (ECACC, Porton Down, UK) was cultured in RPMI 1640 media supplemented with low endotoxin fetal calf serum (10% v/v), 2 mM L-glutamine, 10 units/ml penicillin, and 100 mg/ml streptomycin. Differentiation was induced by treatment (1 x 106 cells/ml) with PMA (20 ng/ml) for 24 h, and after adherent cells were washed and maintained in complete media lacking phenol red.

Preparation and Purification of Human IIA PLA2 Mutants—The preparation of mutations at Val-3 and His-48 have been described previously (16-18). Synthetic complementary oligo-nucleotide primers containing the desired mutation used for the construction of S74C were as follows: S74C forward, 5'-C TCT AAC TCT GGT TGC CGA ATC ACC TGC G-3'; and S74C reverse, 3'-G AGA TTG AGA CCA ACG GCT TAG TGG ACG C-5'. Bacterial protein expression and purification were as described previously (19). Secondary structures were confirmed by CD analysis (18).

Electrospray Ionization Mass Spectrometry—Mass spectrometry was performed on mutant and fluorescently labeled proteins using a Fisons VG QUATTRO II quadrupole mass spectrometer in electrospray mode, and a Micromass LCTTM orthogonal acceleration time of flight mass spectrometer fitted with a nano-electrospray source.

Fluorescent Labeling of Cysteine Mutants—Cysteine mutants were prepared for labeling with thiol-reactive fluorescent dyes by transfer of 0.5 mg of enzyme into 10 mM Tris·HCl, pH 7.4. The enzyme was treated with a 10 M excess of dithiothreitol on ice for 30 min to remove the extra cysteine molecule disulfide bound to the mutant (20). Dithiothreitol was removed after the 30 min of incubation by washing with fresh 10 mM Tris·HCl, pH 7.4, using a concentrator. The sample was analyzed using ESI-MS (described previously) to ensure removal of extra cysteine. The reduced enzyme was treated for 2 h at room temperature in the presence of a 10 M excess of Alexa Fluor-568 maleimide (AF-568) dye in Me2SO, or 50 min on ice in the presence of a 10 M excess of Texas Red-2-sulfonamidoethyl-methanethiosulfonate, 2-[(5-fluorosceinyl)aminocarbonyl]ethyl-methanethiosulfonate, or dansylamidoethyl-methanethiosulfonate dyes. All excess dye was removed through a PD-10 desalting column.

Enzyme Assays—PLA2 measurements were performed using a 11-(dansylamino)undecanoic acid displacement assay (21).

Calcein Release—Calcein release was measured using a Hitachi F2500 fluorescence spectrophotometer (excitation, 490 nm; emission, 420 nm). Data were normalized to 100% release by treatment with 1% Triton X-100 (v/v).

Flow Cytometry—Cells were harvested using Accutase (PAA Laboratories Ltd.) and suspended to 1 x 106 cells/ml. 0.75-ml assays were set up in 1.5-ml microcentrifuge tubes and incubated at 37 °C end over end under desired conditions. After the appropriate time, cells were transferred to FACS tubes and centrifuged at 1000 x g for 5 min to pellet cells. The pellet was washed three times in phosphate-buffered saline, resuspended in 0.5 ml of phosphate-buffered saline, and maintained for flow cytometry (FACSCalibur Flow Cytometer, BD Biosciences), and data were analyzed using Cellquest.

Trypan blue fluorescence quenching was achieved by adding the dye in a 1:1 ratio (by volume) to the sample being analyzed. The difference in fluorescence between samples with and without trypan blue shows the proportion of IIA PLA2 that has been internalized.

Confocal Microscopy—Cells were treated under the desired conditions before being washed in sterile phosphate-buffered saline, and the plasma membranes were stained with concanavalin A-Alexa Fluor-488 for 10 min. Cells were then visualized using a Zeiss confocal microscope (LSM 510 META).

Epifluorescence Microscopy—Cells were cultured at 4 x 104 cells/ml on 13-mm glass coverslips in 24-well plates and treated as above. Coverslips were analyzed using a Nikon Eclipse E800 microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Chemical Labeling of Human Group IIA PLA2 at Position 74 after Cysteine Substitution Mutagenesis Produces Fully Active Proteins—There have been no qualitative or quantitative studies of labeled protein to monitor the binding, potential uptake, and distribution of the human group IIA PLA2 into human cells. To investigate these processes, we used a combination of FACS and fluorescence microscopy. Therefore, it was necessary to produce fluorescent enzyme derivatives and to ensure that attachment per se and position of the fluorescent probe did not affect protein function. Two surface features of the IIA PLA2 protein are important for its potential functions as follows: first, an interfacial (membrane) binding surface surrounding the channel leading to the active site (22), and second, clusters of cationic residues globally distributed over the protein surface capable of binding to HSPGs (22).

Inspection of the IIA PLA2 molecule indicated that amino acid residue Ser-74, on the opposing face from the interfacial binding surface of the protein, was a suitable location for the cysteine substitution (see supplemental Fig. S1). The site-directed mutant, S74C, was produced to provide a chemically reactive thiol group to allow specific labeling. This thiol group was reacted with the methanethiosulfonate derivatives of Texas Red, fluorescein, or dansyl fluorophores or with Alexa Fluor-568 maleimide. The fluorescent derivatives of the catalytically inactive mutant, H48N, were also prepared (H48N,S74C), and the parent active site mutant had <0.5% of the catalytic activity of the wild type enzyme (17). In addition, another mutant, V3C, on the interfacial binding surface was prepared, and both V3C and S74C were labeled with the polarity sensitive dansyl fluorophore (see supplemental Fig. S1). These derivatives will detect binding to phospholipid vesicles that involves both the front and back surface of the protein.

After purification, the identity of each labeled protein was confirmed by ESI-MS (Table 1). To ensure that the labeled enzymes retained functionalities of the parent enzyme, catalytic activities were assayed and were found to be not significantly different. An exception was the dansylated V3C mutant where the interfacial binding surface of the enzyme has been modified. This derivative showed 50% of the parent enzyme activity against DOPG and significantly greater activity against DOPC (Table 2). The enhanced activity with DOPC vesicles as the result of attachment of an aromatic dansyl group at position 3 was not unexpected because tryptophan insertion (V3W) produced increased activity (16) because of enhanced interfacial binding (18). HSPG binding activity, assayed as binding affinity for heparin-Sepharose, was unaffected by all modifications (data not shown). Thus we were confident that when these fluorescent enzymes were added to cultured cells and monitored by FACS and fluorescence microscopy they would mimic the significant properties of the wild type IIA enzyme. Moreover, using a catalytically inactive mutant (Table 2) would clearly define functions of the enzyme that were independent of phospholipid hydrolysis.


View this table:
[in this window]
[in a new window]

  		TABLE 1
ESI-MS analysis of IIA PLA2, mutants, and conjugates

Shown are the predicted masses (daltons) and observed masses.

 


View this table:
[in this window]
[in a new window]

  		TABLE 2
Catalytic activities of human IIA PLA2 and derivatives

The measurements were performed using a fluorescent displacement assay (21). The final concentration of phospholipid was 62.5 µM, and assays were performed in 0.1 M TrisβHCl buffer, pH 8.0, containing 0.1 M NaCl and 1 mM Ca2+.

 
Cell Surface Binding Properties of IIA PLA2—The human monocyte-derived cell line, THP-1, upon differentiation with PMA shows enhanced (>10-fold) HSPG expression (23), and these cells have been widely used to investigate a variety of cellular events (24), particularly phagocytosis and other uptake phenomena (25, 26). Our first objective was to determine whether, and under what conditions, the labeled IIA PLA2 (S74C-Fl) could bind to THP-1 cells. FACS showed that binding to PMA-untreated cells was negligible, but binding was greatly enhanced by prior treatment of cells with PMA for 24 h (Fig. 1A), conditions that produced morphological differentiation and adherence to tissue culture plastic. PMA treatment at 50 ng/ml produced a 14-fold increase in enzyme binding to cells (Fig. 1A) in line with the >10-fold reported increase in HSPG expression (23). Following stimulation by PMA, the binding of the fluorescent enzyme to the cells reached saturation within 60 min (Fig. 1B). The catalytically inactive IIA PLA2 mutant (H48N) showed no significant difference in binding to THP-1 cells compared with the labeled IIA PLA2 (S74C-Fl) protein (Fig. 1C), thus clearly establishing that phospholipid hydrolysis is not a requirement for this additional IIA function.

Binding of IIA PLA2 to Cell Surface PS or HSPGs?—The molecular basis of PMA differentiation leading to binding of IIA PLA2 to THP-1 cells was investigated. Cell differentiation is often associated with increased PS exposure on the cell surface, also a characteristic of cell apoptosis. An increased concentration of this anionic phospholipid in the outer monolayer of the plasma membrane could increase the interfacial binding affinity of cationic IIA PLA2 and enhance membrane hydrolysis (18). PS exposure can be detected using the probe annexin V-AF488 that will bind with high affinity to cell surface PS. Enhanced binding was observed with PMA-differentiated THP-1 cells compared with control cells, although binding was 2-3-fold greater than differentiated cells when apoptosis was induced using etoposide (Fig. 2A). Although annexin V could self-compete, no competition was observed between annexin V and IIA PLA2 (Fig. 2B) suggesting that different cell surface receptors may be involved for the two binding phenomena. Moreover, treatment of cells with 7-ketocholesterol, known to increase surface expression of PS in THP-1 cells, in a dose-dependent manner (27), failed to achieve significant binding of the IIA PLA2 until at high doses (100 µg/ml) that also brought about a concomitant reduction in cell viability (Fig. 2C). Thus our conclusion from these experiments is that PS exposure, at the levels detected following cell differentiation, does not play a major role in the binding of IIA PLA2 to PMA-activated THP-1 cells.

HSPGs have been implicated in the function of IIA PLA2, particularly through the study of the effects of the enzyme expression in transfected cells (13). The role of HSPGs in the binding of IIA PLA2 to THP-1 cells was established by pretreatment of cells for 48 h with chlorate (a competitive inhibitor for the formation of 3'-phosphoadenosine 5'-phosphosulfate, the high energy donor of sulfation reactions). Chlorate treatment resulted in an 80% suppression of the binding of the IIA enzyme. To demonstrate specificity of the chlorate response, cells were given a 24-h recovery period to repopulate HSPGs, and this correlated with a return to IIA binding levels of chlorate-untreated but PMA-differentiated cells (Fig. 3A). Cells remained viable at the concentration of chlorate used. The multivalent electrostatic interaction of proteins with HSPGs is well established, and a similar mode for IIA PLA2 binding was suggested by the dose-dependent inhibition of binding by NaCl, 80% inhibition of binding at 100 mM NaCl. However this did not affect the background fluorescence observed in nondifferentiated cells (Fig. 3B).


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		FIGURE 1.
PMA-dependent binding of active S74C-Fl IIA PLA2 and inactive H48N,S74C-Fl IIA PLA2 to activated THP-1 cells. A, effect of PMA concentration on enzyme binding; cell were pretreated with the indicated concentration of PMA for 24 h followed by treatment with 0.5 µg/ml S74C-Fl. The binding of the fluorescent protein is expressed as % of binding to cells not activated by PMA treatment. B, time course of enzyme binding; cells were untreated (bullet) or pretreated with 20 ng/ml PMA ({circ}) for 24 h followed by addition of 0.5 µg/ml S74C-Fl for the time indicated. The binding of fluorescent protein is expressed asa%of the (background) fluorescence of cells not exposed to fluorescent protein. C, THP-1 cells were pretreated with (gray) or without (black) 20 ng/mlPMA for 24 h followed by incubation with 0.5 µg/ml catalytically active S74C-Fl or catalytically inactive H48N, S74C-Fl for 45 min. Cell-associated fluorescence was determined by FACS analysis and is expressed as % of fluorescence of cells not activated by PMA. Data shown are means ± S.D.

 
Internalization of IIA PLA2 into THP-1 Cells—FACS is unable to discriminate between cell surface and internalized fluorescence from the labeled IIA protein. Cells treated with the membrane impermeable dye trypan blue, after IIA labeling, will have quenched extracellular fluorescence, including that associated with the cell surface. However, trypan blue will not be able to quench internalized fluorescence of viable cells that can be measured (28); this fluorescence will reflect the uptake/internalization of IIA PLA2 by live cells. Using this protocol a time course for the internalization of S74C-Fl into PMA activated THP-1 was determined and compared with nonactivated cells. These results (supplemental Fig. 2) clearly demonstrated internalization of enzyme that approached saturation at 2 h, whereas uptake into nonactivated cells was negligible.

Confocal microscopy also demonstrated internalization (Fig. 4). Uptake of S74C-AF568 (Fig. 4, red) into PMA differentiated THP-1 cells was monitored with time (supplemental Figs. 3-5). The cells were also stained with the plasma membrane marker concanavalinA-AF488 (Fig. 4, green). After 30 min the IIA enzyme was internalized, and AF568 fluorescence was distributed throughout the cytoplasm, whereas localization in the nucleus became apparent later with intense nuclear staining being observed at 90 min (Fig. 4). At the completion of the time course, cell viability had not been significantly affected. However, a few cells became rapidly and intensely stained with enzyme, and such cells appeared to be lacking in concanavalinA-AF488 membrane staining. Possibly these were damaged cells that allowed direct entry of the cationic enzyme, which then bound to anionic surfaces within cells such as anionic phospholipids.

A remarkable characteristic of enzyme uptake was the accompanying and highly significant cell swelling (Table 3; Fig. 4) (see also supplemental Figs. 3-5), a typical characteristic of macropinocytosis (29). Significantly, swelling was also seen with the unlabeled wild type enzyme (supplemental Fig. 6). This important result established that the phenomenon described was a direct consequence of the enzyme uptake and not because of the chemical modification of the enzyme. The wild type enzyme and S74C-AF568 caused a 70% increase in diameter over 90 min, and a similar level of increase was observed with poly-L-arginine. The blocking of IIA PLA2 uptake, by ATP depletion or by treatment with chlorate (Fig. 4D) or ammonium chloride, also blocked the increase in cell size (see below), suggesting that uptake and size increase are linked. Interestingly, the catalytically inactive protein also increased cell size (Fig. 4C); however, the magnitude of increase was less than that observed with the wild type enzyme. These data thus clearly established that uptake and cell swelling (Table 3) do not require phospholipid hydrolysis. Significant uptake and cell swelling were only observed in THP-1 cells that had been activated by PMA (data not shown).


View this table:
[in this window]
[in a new window]

  		TABLE 3
THP-1 cell size and fluorescence uptake quantitation following treatment with IIA PLA2 and the effect of inhibitors

THP-1 cells were exposed to IIA PLA2 for 90 min following prior treatment with PMA except for wild-type enzyme where exposure was for 60 min. The cell diameter of confocal microscopy images was measured for all cells, in a field of view, and % initial cell diameter is shown ± S.D. The theoretical increase in volume assuming a spherical cell of the measured diameter has been calculated. In addition, the effect of pretreatment with inhibitors of IIA uptake on cell diameter prior to exposure to IIA was also monitored for studies involving S74C-AF568. The effect of the polycationic protein poly-L-arginine is also shown. Each cell sample measurement contained > 18 cells in the field of view. All samples with the exception of ATP depletion showed a significant increase in cell size with respect to start sizes (p < 0.001 for all). S74C-AF568 and poly-L-arginine increased cell sizes to levels that were not significantly different from wild-type IIA PLA2. All other treatments showed significant differences for final cell size from wild type (p < 0.028 for all). The extent of size increase for H48N was less and significantly different from wild type (p < 0.028) suggesting a role for phospholipid hydrolysis in the overall process that contributes to cell swelling. The increase in cell fluorescence after 90 min following treatment with fluorescent enzymes is also shown. Values are means ± S.D. (n = 10).

 


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 2.
PS exposure in the outer membrane leaflet of PMA-activated THP-1 cells does not contribute significantly to IIA PLA2 binding. A, cells were untreated or activated with 20 ng/ml PMA for 24 h. Apoptosis was subsequently induced in activated cells by treatment with 100 µM etoposide for 3 h. PS exposure was measured using annexin V-AF488, as described by the manufacturer. B, binding of annexin V-AF488 to activated cell was also measured in the presence of nonfluorescent annexin V or wild type IIA PLA2 at concentrations indicated. Cell-associated fluorescence was determined by FACS analysis. C, THP-1 cells were preincubated with the indicated concentration of 7-ketocholesterol for 24 h. Harvested cells were treated with annexin V-AF488 ({circ}) or 0.25 µg/ml S74C-Fl (bullet) for 45 min. Cell-associated fluorescence was determined by FACS analysis. Cell viability ({blacktriangleup}) was assessed by trypan blue exclusion. Data shown are means ± S.D. A, asterisk indicates significant difference (p < 0.05) to unactivated cells; B, asterisk indicates significant difference (p <0.05) to control cells; C, asterisk indicates significant difference (p < 0.05) between annexin V-AF488 and S74C-FL binding at a given concentration of 7-ketocholesterol.

 


Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 3.
Sodium chlorate and sodium chloride inhibit the binding of IIA PLA2 to PMA-activated THP-1 cells. A, PMA-activated THP-1 cells were untreated, treated for 48 h in the presence of 50 mM sodium chlorate or 50 mM sodium chlorate followed by a 24-h recovery in fresh media in the absence of sodium chlorate. Cells were then incubated with 0.25 µg/ml S74C-Fl for 45 min. *, p < 0.05 compared with untreated cells; **, p < 0.05 compared with chlorate-treated cells. B, THP-1 cells were pretreated with ({circ}) or without (bullet) 20 ng/ml PMA for 24 h. Cells were then incubated with 0.25 µg/ml S74C-Fl for 45 min in the presence of NaCl as indicated. Cell-associated fluorescence was determined by FACS analysis. Data shown are means ± S.D. and asterisk indicates p < 0.05 compared with NaCl untreated.

 
Molecular Basis for the Internalization of IIA PLA2—Following binding to HSPGs, a number of endocytic routes are available to allow internalization of the IIA PLA2. These include clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis (30-35), whereas the uptake route may depend on several features, including cell type and the size, concentration, and charge density of the cargo. However, a common feature is that all endocytic pathways are energy-dependent. The energy dependence of IIA PLA2 internalization was established by using both low temperature and ATP-depletion caused by metabolic inhibitors; both treatments independently resulted in <6% of control uptake. Epifluorescence microscopy of cells treated with S74C-AF568 at 4 °C revealed that the enzyme co-localized with the plasma membrane marker concanavalinA-AF-488 with minimal IIA fluorescence seen within cells compared with treatment at 37 °C (supplemental Fig. 7).


Figure 4
View larger version (175K):
[in this window]
[in a new window]

  		FIGURE 4.
Internalization of IIA PLA2 by PMA-activated THP-1 cells as assessed by confocal microscopy. All images were taken of PMA-activated cells before the addition of enzyme (A), after the addition of S74C-AF568 (0.5 µg/ml) for 90 min (B), and after the addition of catalytically inactive H48N,S74C-AF568 (0.5 µg/ml) for 90 min (C); and cells were pretreated with sodium chlorate (25 mM) for 48 h followed by the addition of S74C-AF568 (0.5 µg/ml) for 90 min (D). Red is S74C-AF568. Green is concanavalin A-AF488. Size marker is 20 µm.

 
A number of inhibitors employed to define the uptake route indicated that the IIA PLA2 uptake was partially inhibited by potassium ion depletion and chlorpromazine addition, treatments that disrupt clathrin-mediated events (Table 4). Similarly cholesterol depletion, by using methyl-β-cyclodextrin (10 mM), resulted in ~70% inhibition of IIA uptake suggesting an involvement of caveolae/lipid rafts (Table 4); however, depletion of membrane cholesterol can also inhibit clathrin-dependent endocytosis and macropinocytosis (36).


View this table:
[in this window]
[in a new window]

  		TABLE 4
Inhibitors of endocytosis reduce internalization of IIA PLA2 into THP-1 cells

Internalization of S74C-AF568 (0.25 µg/ml) by PMA-activated cells was assessed by FACS analysis with trypan blue fluorescence quenching of enzyme that had not been internalized after 45 min. Details of treatments and quenching procedure are described under "Experimental Procedures." Results are presented as percentage control uptake, set at 100%, ±S.D. All treatments showed significant differences from control (p < 0.05) with the exception of potassium ion replenished (NS).

 
Generally, macromolecules that enter cells via the endocytic pathway end up in lysosomes where degradation takes place. However, various cell penetrating peptides avoid degradation, and HIV-TAT, like IIA PLA2, also accumulates in the nucleus (31, 37). Ammonium chloride disrupts endosome acidification and was found to dramatically effect the internalization (Table 4) of IIA PLA2. Thus, lack of acidification could affect release of the IIA PLA2 from an endosomal compartment and/or recycling of endosomes containing HSPGs back to the plasma membrane to allow endocytosis to continue. Ammonium chloride pretreatment also reduced IIA PLA2 induced cell swelling (Table 3).

The considerable cell swelling, described above that accompanies IIA protein uptake, is characteristic of macropinocytosis, a process that involves the uptake of large volumes of fluid into cells. If IIA PLA2 were to enter cells by this route, it would explain the dramatic cell swelling that is observed (Table 3). Cell swelling was fully reversible with time. After 90 min of treatment with IIA PLA2, the enzyme was removed, and cells were observed for a further 24 h. At this time, full recovery was seen with cells returning to their original size. Cell swelling was much reduced in cells when exposed to IIA PLA2 after cholesterol depletion with methyl-β-cyclodextrin, a treatment known to inhibit macropinocytosis.


Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 5.
Bifacial binding of IIA PLA2 to mixed DOPS:DOPC phospholipid vesicles revealed by fluorescence increase of V3C-dansyl and S74C-dansyl IIA derivatives. Mixed SUVs containing molar ratios of DOPS:DOPC (0-100% DOPS) were titrated into 10 mM HEPES, pH 7.5, containing 2 mM EGTA with either 1 nmol of V3C-dansyl (A) or 1 nmol of S74C-dansyl (B). Fluorescence intensity was recorded at 495 nm after excitation at 250 nm. Data shown are means ± S.D. Very similar results were obtained when DOPG was substituted for DOPS. bullet, 100% DOPS; {circ}, 20% DOPS;{blacktriangledown}, 10% DOPS; {Delta}, 5% DOPS; {blacksquare}, 0% DOPS.

 
The accumulated evidence indicates that IIA PLA2 uptake has strong parallels with the numerous studies of the uptake of cationic peptides and proteins, whereas dramatic effects on cell volume, as a result of macropinocytosis, are characteristic of a low specificity, low affinity, high capacity process resulting in a large fluid intake. Consistent with this proposal was the observation that wild type (nonfluorescent) IIA PLA2 failed to compete with the fluorescent enzyme for uptake. Similarly, the cationic peptide, poly-L-arginine, also caused cell swelling (Table 3) but failed to compete with IIA PLA2 binding to the cell (supplemental Fig. 8). It was noted that high concentrations of IIA PLA2, the catalytically inactive mutant, H48N, and poly-L-arginine were toxic to cells, as judged by the failure of such treated cells to exclude trypan blue and the enhanced binding of fluorescent IIA PLA2 to these damaged cells (supplemental Fig. 8).

IIA PLA2-Anionic Phospholipid Vesicles Interaction, Bridging between Anionic Surfaces Produces Cationic Aggregates—The global cationic character of IIA PLA2 with a net charge of +19 is an unusual characteristic of the enzyme and previously linked to its antibacterial properties (6). This characteristic also results in the IIA PLA2 having the unique property of aggregating anionic phospholipid vesicles (38, 39). To explore this phenomenon further, we attached a polarity-sensitive dansyl reporter group to both the interfacial binding surface of the enzyme at position 3 employing a V3C mutant and the reverse face making use of the S74C mutation. The fluorescence of the dansyl group was used to detect membrane binding as a result of desolvation at the membrane-protein interface previously studied using dansylated phospholipid vesicles (40).

The V3C-dansyl and S74C-dansyl proteins both bound to phospholipid vesicles containing increasing proportions of anionic phospholipid (Fig. 5). This showed that the enzyme was able to bind to vesicles by both the front and back faces of the protein and could therefore bridge between anionic particles. Binding was observed at as low as 5 mol % anionic phospholipids, DOPG or DOPS, although no binding was observed to vesicles containing only DOPC. Saturation was achieved at a phospholipid:protein ratio of ~10:1 in vesicles containing 100% anionic phospholipid, and under such conditions the overall complex was calculated to be positively charged because each IIA protein molecule has a charge of + 19.

Light scattering studies also showed that aggregation was produced on addition of the IIA protein using vesicles containing as low as 5 mol % anionic phospholipid (Fig. 6). Aggregation was achieved in the absence of calcium ions to prevent phospholipid hydrolysis. The extent of light scattering increased as the mol % of anionic phospholipids increased; however, with 100% anionic phospholipids less light scattering/aggregation was observed than with 5% anionic phospholipids. To validate the integrity of vesicles treated with the IIA protein, they were pre-loaded with calcein, release of which would suppress the self-quenching and produce a large increase in fluorescence intensity. Calcein release only occurred when the IIA enzyme bound to 100% DOPG vesicles and was not observed with the pancreatic PLA2 (Fig. 7), whereas vesicles containing up to 20 mol % PS or phosphatidylglycerol released negligible calcein and were therefore assumed to remain intact. The lower level of aggregation seen with vesicles containing 100 mol % anionic phospholipid (Fig. 6) may be due to vesicle disruption, as such vesicles released calcein.

Overall these physical data established that the IIA enzyme was uniquely able to bind to anionic phospholipid surfaces involving at least the front and back face of the enzyme, producing aggregates that, based on binding stoichiometry, would be positively charged. Such aggregation was observed at 5-20 mol % PS, the range of concentration of exposed anionic phospholipid found in cell debris and microparticles. However the precise structure of supramolecular aggregates is not known (38, 39) and would probably vary depending on the nature of the anionic target.

We questioned whether a unique role for the IIA enzyme in vivo was to coat and aggregate anionic cell debris thus allowing its uptake into cells prior to degradation. If this were the case then the uptake of anionic phospholipid vesicles into cells should be enhanced by the presence of the IIA PLA2 to facilitate interactions with cell surface HSPGs and endocytosis. To test this hypothesis fluorescently labeled aggregates were prepared containing 20 mol % PS, 75 mol % phosphatidylcholine together with 5 mol % PE labeled with Texas Red. The uptake of these vesicles into cells was monitored by FACS (Fig. 8) and confocal microscopy (Fig. 9). Interestingly, significant uptake was only observed after extensive prior washing of cells (Fig. 8A). We interpret this observation as a requirement to remove endogenous competing unlabeled debris that accumulates in the medium as a result of cell dispersal from monolayer cultures. Under these stringent washing conditions, a >10-fold enhancement of vesicle uptake was observed using FACS, in the presence of IIA PLA2 or the noncatalytic H48N mutant, whereas no uptake of zwitterionic vesicles devoid of anionic phospholipid was observed (Fig. 8B). Confocal analysis confirmed these data with fluorescence being distributed throughout the cell cytoplasm. Unlike the uptake of fluorescent IIA PLA2, no obvious nuclear localization of the fluorescent phospholipid was detected (Fig. 9); however, cell swelling was observed suggesting dissociation of vesicle and IIA PLA2 in the cytosolic or endocytic compartment prior to nuclear localization of IIA PLA2.


Figure 6
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 6.
DOPG concentration influences the extent of aggregation of mixed DOPG vesicles on the addition of IIA PLA2. Fluorescent light scatter was recorded over time with excitation and emission set at 505 nm. Light scatter increased after the addition of 1 µM IIA PLA2 (0% DOPG = 100% DOPC) for mixed DOPG:DOPC SUVs, as indicated and in the absence of calcium ions up to 20% DOPG. 100% DOPG showed a reduced level of light scatter attributed to an effect of IIA PLA2 on vesicle integrity (see Fig. 7).

 


Figure 7
View larger version (12K):
[in this window]
[in a new window]

  		FIGURE 7.
IIA PLA2 phospholipase activity-independent disruption of vesicles containing 100% DOPG. Calcein-loaded mixed DOPG:DOPC SUVs as indicated (10 µM) were treated with 1 µM of either IIA PLA2 or the catalytically inactive mutant H48N, S74C, or porcine pancreatic PLA2 in the absence of calcium ions. Calcein release leading to suppression of autoquenching was measured by the increase in fluorescence at 490 nm following excitation at 420 nm. Data were normalized to 100% calcein release with 1% Triton X-100. Data shown are means ± S.D. Asterisk indicates significant difference (p < 0.05) to 100% DOPG with IIA PLA2.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
There are at least 10 isoforms of human secreted PLA2, and the physiological roles of these enzymes remain obscure (41), but the roles must, at least in part, reflect structural and functional differences resulting from the different surface properties of the enzymes (42). The IIA PLA2 is an extracellular acute phase protein first observed at high concentrations in the synovial fluid of patients with rheumatoid arthritis (1), and the primary protein sequence was determined following purification from both synovial fluid and platelets (43). It has a clear involvement in the inflammatory response, as its synthesis and secretion are under the control of inflammatory cytokines. Serum levels can rise up to 1000-fold, in severe inflammatory conditions such as sepsis, to >1 µg/ml (44, 45), whereas the concentration of enzyme in human tears is normally ~30 µg/ml (46). Interestingly, human corneal epithelial cells appear not to be affected by the IIA PLA2 with essentially no phospholipid hydrolysis being observed (39) and suggesting a lack of interaction between IIA and these cells. Physiologically relevant concentrations of 0.25-0.5 µg/ml of enzyme were used for the majority of studies described in this paper.

The enzyme has certain unique features compared with other human secreted PLA2s; it is highly cationic with an overall charge of +19 involving arginine and lysine residues globally distributed over the protein surface (47). The enzyme binds with high affinity to anionic phospholipid interfaces, but it has a very low affinity for zwitterionic interfaces (characteristic of the external monolayer of the plasma membrane); and as a result such phospholipid membranes are not hydrolyzed. In vivo this IIA protein is produced in large amounts compared with the other human nonpancreatic secreted PLA2s, particularly in response to inflammation. Together we believe these characteristics reflect physiological functions.


Figure 8
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 8.
Exogenously added IIA PLA2 enhances the internalization of DHPE-Texas Red-labeled DOPG vesicles by PMA-activated THP-1 cells. PMA-activated THP-1 cells were incubated for 60 min with 1800 nM labeled phospholipid vesicles in the presence of H48N,S74C IIA PLA2 at the concentrations indicated. A, vesicles consisted of 20% DOPG, 75% DOPC, 5% DHPE-Texas Red. Cells were washed twice ({circ}) or five times (bullet) with phosphate-buffered saline. B, vesicles consisted of 20% DOPG, 75% DOPC, 5% DHPE-Texas Red ({circ}) or 95% DOPC, 5% DHPE-Texas Red (bullet). Cells were washed five times. The internalization of fluorescence was assessed by FACS analysis following trypan blue quenching of external fluorescence. Data shown are means ± S.D. A, asterisk indicates significant difference (p < 0.05) between two and five washes at a given concentration of H48N,S74C-Fl; B, asterisk indicates significant difference (p < 0.05) between 20 and 0% DOPG-containing vesicles at a given concentration of H48N,S74C-Fl.

 


Figure 9
View larger version (109K):
[in this window]
[in a new window]

  		FIGURE 9.
Confocal microscopy analysis of the internalization of DHPE-Texas Red-labeled DOPG vesicles by THP-1 cells. PMA-activated THP-1 cells were incubated for 60 min with 1800 nM labeled phospholipid vesicles in the presence of no IIA PLA2 (A), 2 µg/ml IIA PLA2 (B), or 2 µg/ml H48N PLA2 (C). Green is concanavalin A-AF488 (panel I). Red is DHPE-Texas Red (panel II). Panel III in each case is the merged channels. Size marker is 20 µm.

 
The enzyme has an important antibacterial role as part of innate immunity through its phospholipase activity (3-5) and is able to selectively target Gram-positive bacteria such as Staphylococcus aureus. The global positive charge on the protein surface is essential for the penetration of the anionic bacterial cell wall, whereas high affinity binding to the anionic cell membrane results in phospholipid hydrolysis and assists in bacterial killing (6). Gram-negative bacteria may also be targeted, but other factors such as bactericidal/permeability-increasing protein are also required to permeabilize the lipopolysaccharide coat and allow access of the enzyme to the cell membrane (48).

This study identifies four important new properties of human IIA PLA2 that are independent of phospholipase activity and linked to the effects of the enzyme on host cell function. First, the enzyme is taken up by activated human THP-1 cells in an energy-dependent process requiring HSPGs and involving macropinocytosis. Second, uptake does not require the enzyme to be catalytically active, as these properties were also demonstrated by a catalytically inactive mutant H48N. Third, confocal microscopy reveals that the enzyme accumulates in the nucleus, a characteristic also seen by other cationic cell-penetrating proteins and peptides. Fourth, the enzyme is able to bind to and aggregate anionic phospholipid vesicles and enhance their uptake. This observation suggests a scavenging role for the protein in removing anionic cell debris.

Cellular Binding and Uptake—Much research effort has been directed toward investigating the interaction between secreted PLA2s and the plasma membrane (14, 15); however, to date no human high affinity membrane receptor has been reported for the human IIA PLA2. Moreover, the enzyme shows very low affinity for the zwitterionic plasma membrane consistent with the negligible rates of phospholipid hydrolysis for such substrates (39, 42, 49). PS exposure, as a result of cell apoptosis, could facilitate interfacial binding and hydrolysis (11), but the physiological relevance for IIA participation in this process remains to be established. The binding of the IIA enzyme to cell surface HSPGs is well established, but the physiological role of this process is not clear (13); however, membrane hydrolysis is not an outcome (50).

We have shown, using fluorescently labeled human IIA enzyme, cell surface binding of IIA to HSPGs of THP-1 cells and internalization in an energy-dependent process. Uptake requires that THP-1 cells must first be differentiated by prior treatment with PMA, a process in which they become adherent and develop a macrophage-like phenotype. Minimal binding was observed by IIA PLA2 with nonactivated cells. Interestingly, PMA treatment dramatically enhances the cell surface expression of HSPGs in THP-1 cells (23). A remarkable feature of binding and uptake was the associated cell swelling that was dramatic but fully reversible with time. No cell swelling was observed in nonactivated THP-1 cells or in cells pretreated with chlorate to inhibit the sulfation of HSPGs that prevented IIA PLA2 uptake. Cell swelling seemed to be a general response to cationic proteins and was also seen with poly-L-arginine. Cell swelling is probably due to excessive fluid uptake and its acute retention through macropinocytosis.

Phospholipid Hydrolysis Is Not Required for IIA PLA2 Function Indicating an Accessory Role for the Enzyme—The active site of secreted PLA2s is characterized by an essential histidine residue. Substituting it for asparagine (H48N) produces a protein with <0.5% of the catalytic activity of the wild type enzyme (17). A fluorescent derivative of this mutant protein revealed that binding and uptake, including cell swelling, were essentially unaffected by the loss of catalytic activity, although the level of swelling appeared reduced compared with wild type. Thus phospholipid hydrolysis and the release of potential lipid mediators, fatty acids, and lysophospholipid, do not appear to play a significant role in the uptake process into THP-1 cells described here. In contrast, in other studies binding of IIA to HEK293 cells was observed, but only in a double-tryptophan mutant that allowed binding to the zwitterionic plasma membrane and subsequent membrane hydrolysis (18). Using another active site mutant, H48Q, it was observed that the autocrine expression of IIA in mesangial cells, by added IIA, did require the enzyme to be catalytically active. One notion was that active IIA provided ligands for peroxisome proliferator-activated receptor {alpha} activation as part of the regulation of gene expression (51). Thus the observations described in this study probably reflect cell type and cell differentiation-specific responses, and it is well established that cell surface HSPG expression plays a major role in cell function (52). An accessory, noncatalytic role is seen with other proteins, for example the role of cytochrome c in apoptosis.

Nuclear Accumulation of IIA PLA2—Confocal microscopy studies with the fluorescent IIA PLA2 and its catalytically inactive mutant have clearly established that after uptake IIA PLA2 is targeted to the nucleus. The enzyme survives endocytic uptake, escapes from the endocytic route, and is released into the cytoplasm without proteolysis. This is a different outcome to that seen when the rat IIA enzyme is taken up by rat mast cells where protein degradation ensues (53), and thus mast cells may be considered as an end point for IIA PLA2 in inflammation. The highly cationic nature of the IIA protein could facilitate nuclear localization, a characteristic seen with other cell-penetrating cationic proteins (37). How this IIA enzyme can affect nuclear function in THP-1 cells is not known at this time. We have preliminary evidence that COX-2 expression is increased further in lipopolysaccharide-primed THP-1 cells by both wild type and the catalytically inactive mutant of IIA PLA2; however, the mechanism for gene regulation in this system is unknown, although there is an extensive literature on the post-transcriptional regulation of COX-2.

Vesicle Aggregation and the Removal of Cell Debris—A role of IIA PLA2 in removing cell debris has been previously advocated involving membrane hydrolysis and another acute phase protein, C-reactive protein (12), whereas the hydrolysis of phospholipid microparticles to generate bioactive lipid mediators, such as lysophosphatidic acid, has been proposed (54). Inherent in both these proposals is the hydrolysis of membrane phospholipids. Of particular interest is the unique ability of the PLA2 to bind to and aggregate anionic phospholipid vesicles (38, 39). We have confirmed that this phenomenon does not require the enzyme to be catalytically active and occurs with vesicles containing only 5 mol % anionic phospholipid (PS or phosphatidylglycerol). Using calcein-loaded vesicles, we established that the vesicles remained structurally intact on aggregation except for the unphysiological situation where the vesicles are prepared with 100% anionic phospholipid. When titrating onto the vesicle surface was monitored using fluorescent reporter groups attached to the IIA protein, a molar ratio of about 10:1 was indicated between phospholipid and protein. Although the precise physical nature of any phospholipid IIA aggregate remains to be established (38, 39), such aggregates would maintain an overall positive charge due to the high positive charge on the IIA protein molecule.

The IIA protein could have a role in aggregating anionic phospholipid debris, including microparticles, producing positively charged complexes that are taken up and degraded by cells expressing HSPGs. By using fluorescently labeled vesicles, we demonstrated that the cellular uptake of such vesicles is greatly enhanced by the IIA PLA2 in a process that does not require the enzyme to be catalytically active. It is presumed that the phospholipid debris is degraded by hydrolases within a lysosomal compartment, whereas the cationic IIA protein is able to escape such degradation allowing nuclear localization.

Debris does accumulate under inflammatory conditions, such as during tissue trauma and infection, and large amounts of the IIA PLA2 are produced. The original discovery of the enzyme in the synovial fluid of patients with rheumatoid arthritis may reflect the need to remove inflammatory debris from joint fluid. A model illustrating the concept of debris coating by IIA PLA2, bridging to cell surface-expressed HSPG and subsequent cellular uptake, is proposed (Fig. 10).

This model takes into account the variety of uptake routes that have been proposed for cationic proteins and peptides, including those associated with liposomes (55). These proposed uptake routes are clathrin-mediated endocytosis, caveolae/lipid raft-mediated endocytosis, and macropinocytosis. An emerging feature is that uptake route reflects the physical nature of the aggregate and the HSPGs expressed on the cell surface. The uptake of cationic proteins and peptides plus associated cargo into cells is an active research area as already highlighted (29-35) and has been reviewed recently (56-58).

The wider implications of the proposed IIA PLA2-mediated cell debris removal must be considered. Loss of membrane asymmetry is usually accompanied by the shedding of microparticles. Microparticles have been implicated in cardiovascular disease (59), arthritis (60), and also cancer (61); their removal may be critical (62). It should be noted for example that elevated IIA PLA2 expression was identified as being protective against gastric cancer (63, 64). Microparticles released from various cells generated during inflammation have also been implicated in transferring host protein antigens between cells. Of particular interest is the uptake by neutrophils of microparticles derived from activated platelets (65); platelet activation results in the release of IIA PLA2 (43). We also believe that this system of cationic aggregate uptake and nuclear trafficking is the basis of nonviral gene transfection procedure using cationic liposomes and other delivery vehicles. These transfection protocols may have hijacked the normal system that allows cells to remove large amounts of anionic extracellular debris, including DNA released from damaged cells. Certainly DNA transfection relies on cell surface HSPGs (66), whereas macropinocytosis has also been implicated (55).


Figure 10
View larger version (44K):
[in this window]
[in a new window]

  		FIGURE 10.
Model for the IIA PLA2 uptake into cells and role in uptake of anionic particles. In this model the globally cationic IIA PLA2 molecule is able to interact directly with cell surface HSPGs or to produce cationic aggregates as a result of multiple interactions with anionic particles. The nature of the aggregate will depend on the size and charge density of the particle, and this may also determine the endocytic pathway that is used. Following endocytosis, acidification and possibly other endosomal events should allow IIA PLA2 release and nuclear accumulation.

 
In summary, we have described a novel and noncatalytic accessory role for human IIA PLA2 in the scavenging and bulk removal of anionic debris involving macropinocytosis. The precise pathway for debris hydrolysis within the cell and the effects of nuclear targeting of the IIA protein on gene expression/cell phenotype remain to be determined. An involvement of IIA in microparticle uptake may reflect more specific physiological roles. Thus, the IIA PLA2 may have primarily an anti-inflammatory (resolving) role in the inflammatory response, but it may also communicate with the adaptive immune response through, for example, delivering foreign antigens to antigen-presenting cells.


   FOOTNOTES
 
* This work was supported by a studentship (to C. N. B.) from the Biotechnology and Biological Sciences Research Council (UK). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S8. Back

1 To whom correspondence should be addressed: School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, SO16 7PX, UK. Tel.: 44-2380594308; Fax: 44-2380594459; E-mail: dcw{at}soton.ac.uk.

2 The abbreviations used are: IIA PLA2, human group IIA secreted phospholipase A2; FACS, fluorescence-activated cell sorting; S74C-Fl, human group IIA PLA2 mutant labeled with fluorescein at position 74; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; S74C-AF568, human group IIA PLA2 mutant labeled with Alexa Fluor-568 at position 74; V3C-dansyl, human group IIA PLA2 mutant labeled with dansyl at position 3; S74C-dansyl, human group IIA PLA2 mutant labeled with dansyl at position 74; ESI-MS, electrospray ionization mass spectrometry; DOPG, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPS, 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]; PS, phosphatidylserine; HSPG, heparan sulfate proteoglycan; PMA, phorbol 12-myristate 13-acetate; SUV, small unilamellar vesicle; DHPE, 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine. Back


   ACKNOWLEDGMENTS
 
We thank Dr. A. Buckland and Dr. S. Edwards for preliminary work in the preparation of IIA PLA2 mutants. We are very grateful to Dr. N. Smyth for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pruzanski, W., Vadas, P., Stefanski, E., and Urowitz, M. B. (1985) J. Rheumatol. 12, 211-216[Medline] [Order article via Infotrieve]
  2. Crowl, R. M., Stoller, T. J., Conroy, R. R., and Stoner, C. R. (1991) J. Biol. Chem. 266, 2647-2651[Abstract/Free Full Text]
  3. Wright, G. W., Ooi, C. E., Weiss, J., and Elsbach, P. (1990) J. Biol. Chem. 265, 6675-6681[Abstract/Free Full Text]
  4. Buckland, A. G., and Wilton, D. C. (2000) Biochim. Biophys. Acta 1488, 71-82[Medline] [Order article via Infotrieve]
  5. Femling, J. K., Nauseef, W. M., and Weiss, J. P. (2005) J. Immunol. 175, 4653-4661[Abstract/Free Full Text]
  6. Beers, S. A., Buckland, A. G., Koduri, R. S., Cho, W., Gelb, M. H., and Wilton, D. C. (2002) J. Biol. Chem. 277, 1788-1793[Abstract/Free Full Text]
  7. Murakami, M., and Kudo, I. (2001) Adv. Immunol. 77, 163-194[Medline] [Order article via Infotrieve]
  8. Kudo, I., and Murakami, M. (2002) Prostaglandins Other Lipid Mediators 68, 3-58[CrossRef][Medline] [Order article via Infotrieve]
  9. Wilton, D. C. (2005) Eur. J. Lipid Sci. Technol. 107, 193-205[CrossRef]
  10. Triggiani, M., Granata, F., Giannattasio, G., and Marone, G. (2005) J. Allergy Clin. Immunol. 116, 1000-1006[CrossRef][Medline] [Order article via Infotrieve]
  11. Atsumi, G., Murakami, M., Tajima, M., Shimbara, S., Hara, N., and Kudo, I. (1997) Biochim. Biophys. Acta 1349, 43-54[Medline] [Order article via Infotrieve]
  12. Hack, C. E., Wolbink, G. J., Schalkwijk, C., Speijer, H., Hermens, W. T., and vandenBosch, H. (1997) Immunol. Today 18, 111-115[CrossRef][Medline] [Order article via Infotrieve]
  13. Murakami, M., Koduri, R. S., Enomoto, A., Shimbara, S., Seki, M., Yoshihara, K., Singer, A., Valentin, E., Ghomashchi, F., Lambeau, G., Gelb, M. H., and Kudo, I. (2001) J. Biol. Chem. 276, 10083-10096[Abstract/Free Full Text]
  14. Lambeau, G., and Lazdunski, M. (1999) Trends Pharmacol. Sci. 20, 162-170[CrossRef][Medline] [Order article via Infotrieve]
  15. Rouault, M., Le Calvez, C., Boilard, E., Surrel, F., Singer, A., Ghomashchi, F., Bezzine, S., Scarzello, S., Bollinger, J., Gelb, M. H., and Lambeau, G. (2007) Biochemistry 46, 1647-1662[CrossRef][Medline] [Order article via Infotrieve]
  16. Baker, S. F., Othman, R., and Wilton, D. C. (1998) Biochemistry 37, 13203-13211[CrossRef][Medline] [Order article via Infotrieve]
  17. Edwards, S. H., Thompson, D., Baker, S. F., Wood, S. P., and Wilton, D. C. (2002) Biochemistry 41, 15468-15476[CrossRef][Medline] [Order article via Infotrieve]
  18. Beers, S. A., Buckland, A. G., Giles, N., Gelb, M. H., and Wilton, D. C. (2003) Biochemistry 42, 7326-7338[CrossRef][Medline] [Order article via Infotrieve]
  19. Othman, R., Baker, S., Li, Y., Worrall, A. F., and Wilton, D. C. (1996) Biochim. Biophys. Acta 1303, 92-102[Medline] [Order article via Infotrieve]
  20. Lin, Y., Nielsen, R., Murray, D., Hubbell, W. L., Mailer, C., Robinson, B. H., and Gelb, M. H. (1998) Science 279, 1925-1929[Abstract/Free Full Text]
  21. Wilton, D. C. (1990) Biochem. J. 266, 435-439[Medline] [Order article via Infotrieve]
  22. Snitko, Y., Koduri, R. S., Han, S. K., Othman, R., Baker, S. F., Molini, B. J., Wilton, D. C., Gelb, M. H., and Cho, W. H. (1997) Biochemistry 36, 14325-14333[CrossRef][Medline] [Order article via Infotrieve]
  23. Edwards, I. J., Xu, H. Z., Obunike, J. C., Goldberg, I. J., and Wagner, W. D. (1995) Arterioscler. Thromb. Vasc. Biol. 15, 400-409[Abstract/Free Full Text]
  24. Abe, A., Poucher, H. K., Hiraoka, M., and Shayman, J. A. (2004) J. Lipid Res. 45, 667-673[Abstract/Free Full Text]
  25. Berges, C., Naujokat, C., Tinapp, S., Wieczorek, H., Hoh, A., Sadeghi, M., Opelz, G., and Daniel, V. (2005) Biochem. Biophys. Res. Commun. 333, 896-907[CrossRef][Medline] [Order article via Infotrieve]
  26. Makoveichuk, E., Castel, S., Vilaro, S., and Olivecrona, G. (2004) Biochim. Biophys. Acta 1686, 37-49[Medline] [Order article via Infotrieve]
  27. Berthier, A., Lemaire-Ewing, S., Prunet, C., Montange, T., Vejux, A., Pais De Barros, J. P., Monier, S., Gambert, P., Lizard, G., and Néel, D. (2005) FEBS J. 272, 3093-3104[CrossRef][Medline] [Order article via Infotrieve]
  28. Loike, J. D., and Silverstein, S. C. (1983) J. Immunol. Methods 57, 373-379[CrossRef][Medline] [Order article via Infotrieve]
  29. Norbury, C. C. (2006) Immunology 117, 443-451[CrossRef][Medline] [Order article via Infotrieve]
  30. Kawamura, K. S., Sung, M., Bolewska-Pedyczak, E., and Py, J. (2006) Biochemistry 45, 1116-1127[CrossRef][Medline] [Order article via Infotrieve]
  31. Melikov, K., and Chernomordik, L. V. (2005) Cell. Mol. Life Sci. 62, 2739-2749[CrossRef][Medline] [Order article via Infotrieve]
  32. Kaplan, I. M., Wadia, J. S., and Dowdy, S. F. (2005) J. Controlled Release 102, 247-253[CrossRef][Medline] [Order article via Infotrieve]
  33. Falcone, S., Cocucci, E., Podini, P., Kirchhausen, T., Clementi, E., and Meldolesi, J. (2006) J. Cell Sci. 119, 4758-4769[Abstract/Free Full Text]
  34. Nakase, I., Tadokoro, A., Kawabata, N., Takeuchi, T., Katoh, H., Hiramoto, K., Negishi, M., Nomizu, M., Sugiura, Y., and Futaki, S. (2007) Biochemistry 46, 492-501[CrossRef][Medline] [Order article via Infotrieve]
  35. Duchardt, F., Fotin-Mleczek, M., Schwarz, H., Fischer, R., and Brock, R. (2007) Traffic 8, 848-866[CrossRef][Medline] [Order article via Infotrieve]
  36. Subtil, A., Gaidarov, I., Kobylarz, K., Lampson, M. A., Keen, J. H., and Mcgraw, T. E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6775-6780[Abstract/Free Full Text]
  37. Vives, E., Brodin, P., and Lebleu, B. (1997) J. Biol. Chem. 272, 16010-16017[Abstract/Free Full Text]
  38. Canaan, S., Nielsen, R., Ghomashchi, F., Robinson, B. H., and Gelb, M. H. (2002) J. Biol. Chem. 277, 30984-30990[Abstract/Free Full Text]
  39. Bezzine, S., Bollinger, J. G., Singer, A. G., Veatch, S. L., Keller, S. L., and Gelb, M. H. (2002) J. Biol. Chem. 277, 48523-48534[Abstract/Free Full Text]
  40. Jain, M. K., and Vaz, W. C. (1987) Biochim. Biophys. Acta 905, 1-8[Medline] [Order article via Infotrieve]
  41. Schaloske, R. H., and Dennis, E. A. (2006) Biochim. Biophys. Acta 1761, 1246-1259[Medline] [Order article via Infotrieve]
  42. Singer, A. G., Ghomashchi, F., Le Calvez, C., Bollinger, J., Bezzine, S., Rouault, M., Sadilek, M., Nguyen, E., Lazdunski, M., Lambeau, G., and Gelb, M. H. (2002) J. Biol. Chem. 277, 48535-48549[Abstract/Free Full Text]
  43. Kramer, R. M., Hession, C., Johansen, B., Hayes, G., McGray, P., Chow, E. P., Tizard, R., and Pepinsky, R. B. (1989) J. Biol. Chem. 264, 5768-5775[Abstract/Free Full Text]
  44. Groeneveld, A. B. J., Tacx, A. N., Bossink, A. W. J., Van Mierlo, G. J., and Hack, C. E. (2003) Clin. Immunol. 106, 106-115[CrossRef][Medline] [Order article via Infotrieve]
  45. Nevalainen, T. J., Eerola, L. I., Rintala, E., Jukka, V., Laine, O., Lambeau, G., and Timo, J. N. A. (2005) Biochim. Biophys. Acta 1733, 210-223[Medline] [Order article via Infotrieve]
  46. Qu, X. D., and Lehrer, R. I. (1998) Infect. Immun. 66, 2791-2797[Abstract/Free Full Text]
  47. Scott, D. L., Mandel, A. M., Sigler, P. B., and Honig, B. (1994) Biophys. J. 67, 493-504[Medline] [Order article via Infotrieve]
  48. Elsbach, P., and Weiss, J. (1998) Curr. Opin. Immunol. 10, 45-49[CrossRef][Medline] [Order article via Infotrieve]
  49. Bezzine, S., Koduri, R. S., Valentin, E., Murakami, M., Kudo, I., Ghomashchi, F., Sadilek, M., Lambeau, G., and Gelb, M. H. (2000) J. Biol. Chem. 275, 3179-3191[Abstract/Free Full Text]
  50. Koduri, R. S., Baker, S. F., Snitko, Y., Han, S. K., Cho, W. H., Wilton, D. C., and Gelb, M. H. (1998) J. Biol. Chem. 273, 32142-32153[Abstract/Free Full Text]
  51. Beck, S., Lambeau, G., Scholz-Pedretti, K., Gelb, M. H., Janssen, M. J. W., Edwards, S. H., Wilton, D. C., Pfeilschifter, J., and Kaszkin, M. (2003) J. Biol. Chem. 278, 29799-29812[Abstract/Free Full Text]
  52. Parish, C. R. (2006) Nat. Rev. Immunol. 6, 633-643[CrossRef][Medline] [Order article via Infotrieve]
  53. Enomoto, A., Murakami, M., and Kudo, I. (2000) Biochem. Biophys. Res. Commun. 276, 667-672[CrossRef][Medline] [Order article via Infotrieve]
  54. Fourcade, O., Simon, M. F., Viode, C., Rugani, N., LeBalle, F., Ragab, A., Fournie, B., Sarda, L., and Chap, H. (1995) Cell 80, 919-927[CrossRef][Medline] [Order article via Infotrieve]
  55. Khalil, I. A., Kogure, K., Futaki, S., and Harashima, H. (2006) J. Biol. Chem. 281, 3544-3551[Abstract/Free Full Text]
  56. Jones, A. T. (2007) J. Cell. Mol. Med. 11, 670-684[CrossRef][Medline] [Order article via Infotrieve]
  57. Futaki, S., Nakase, I., Taclokoro, A., Takeuchi, T., and Jones, A. T. (2007) Biochem. Soc. Trans. 35, 784-787[CrossRef][Medline] [Order article via Infotrieve]
  58. Poon, G. M. K., and Gariepy, J. (2007) Biochem. Soc. Trans. 35, 788-793[CrossRef][Medline] [Order article via Infotrieve]
  59. VanWijk, M. J., VanBavel, E., Sturk, A., and Nieuwland, R. (2003) Cardiovasc. Res. 59, 277-287[Abstract/Free Full Text]
  60. Distler, J. H. W., Jungel, A., Huber, L. C., Seemayer, C. A., Reich, C. F., Gay, R. E., Michel, B. A., Fontana, A., Gay, S., Pisetsky, D. S., and Distler, O. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 2892-2897[Abstract/Free Full Text]
  61. Janowska-Wieczorek, A., Wysoczynski, M., Kijowski, J., Marquez-Curtis, L., Machalinski, B., Ratajczak, J., and Ratajczak, M. Z. (2005) Int. J. Cancer 113, 752-760[CrossRef][Medline] [Order article via Infotrieve]
  62. Kim, H. K., Song, K. S., Park, Y. S., Kang, Y. H., Lee, Y. J., Lee, K. R., Kim, H. K., Ryu, K. W., Bae, J. M., and Kim, S. (2003) Eur. J. Cancer 39, 184-191[CrossRef][Medline] [Order article via Infotrieve]
  63. Leung, S. Y., Chen, X., Chu, K. M., Yuen, S. T., Mathy, J., Ji, J. F., Chan, A. S. Y., Li, R., Law, S., Troyanskaya, O. G., Tu, I. P., Wong, J., So, S., Botstein, D., and Brown, P. O. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 16203-16208[Abstract/Free Full Text]
  64. Aggarwal, A., Guo, D. L., Hoshida, Y., Yuen, S. T., Chu, K. M., So, S., Boussioutas, A., Chen, X., Bowtell, D., Aburatani, H., Leung, S. Y., and Tan, P. (2006) Cancer Res. 66, 232-241[Abstract/Free Full Text]
  65. Salanova, B., Choi, M., Rolle, S., Wellner, M., Luft, F. C., and Kettritz, R. (2007) J. Biol. Chem. 282, 27960-27969[Abstract/Free Full Text]
  66. Mislick, K. A., and Baldeschwieler, J. D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12349-12354[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
283/8/5034    most recent
M708844200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Birts, C. N.
Right arrow Articles by Wilton, D. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Birts, C. N.
Right arrow Articles by Wilton, D. C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2008 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement