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Article |
Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor
Correspondence to B. Engelmann: Bernd.Engelmann{at}med.uni-muenchen.de
Although their contribution remains unclear, lipids may facilitate noncanonical routes of protein internalization into cells such as those used by cell-penetrating proteins. We show that protein C inhibitor (PCI), a serine protease inhibitor (serpin), rapidly transverses the plasma membrane, which persists at low temperatures and enables its nuclear targeting in vitro and in vivo. Cell membrane translocation of PCI necessarily requires phosphatidylethanolamine (PE). In parallel, PCI acts as a lipid transferase for PE. The internalized serpin promotes phagocytosis of bacteria, thus suggesting a function in host defense. Membrane insertion of PCI depends on the conical shape of PE and is associated with the formation of restricted aqueous compartments within the membrane. Gain- and loss-of-function mutations indicate that the transmembrane passage of PCI requires a branched cavity between its helices H and D, which, according to docking studies, precisely accommodates PE. Our findings show that its specific shape enables cell surface PE to drive plasma membrane translocation of cell-penetrating PCI.
B. Engelmann's present address is Institut für Klinische Chemie, Ludwig-Maximilians-Universität München, 81377 Munich, Germany.
Abbreviations used in this paper: CF, carboxyfluorescein; GUV, giant unilamellar vesicles; HC-II, heparin cofactor II; LUV, large unilamellar vesicles; NMR, nuclear magnetic resonance; PC, phosphatidylcholine; PCI, protein C inhibitor; PE, phosphatidylethanolamine; Pr3+, praseodymium; PS, phosphatidylserine; rPCI, recombinant PCI; SUV, small unilamellar vesicles; uPCI, urinary PCI.
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Introduction |
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Distinctive properties of the lipid architecture of the cell membrane include lateral microdomains enriched in cholesterol and (glyco)sphingolipids, which constitute a platform for signal transmission and the internalization of microorganisms (rafts and caveolae; Simons and Vaz, 2004; Jacobson et al., 2007). Another key feature of the plasma membrane is represented by the transverse asymmetry of lipids. Under specific biological conditions, this asymmetry is attenuated, as exemplified by the surface exposure of phosphatidylserine (PS), which provides a recognition signal for the phagocytosis of apoptotic cells (Gardai et al., 2006) and establishes a catalytic surface for proteases implicated in blood coagulation (Bevers et al., 1999). Under the same conditions, the exposure of phosphatidylethanolamine (PE) is enhanced (Emoto et al., 1997; Smirnov et al., 1999). However, in contrast to the well-known functions of PS exposure, the functional meaning of PE externalization is largely unknown.
We found that the cellular internalization of the serpin protein C inhibitor (PCI) is crucially supported by plasma membrane PE, which enables its rapid targeting to the nucleus both in vitro and in vivo. Our findings position PCI as an eminent candidate for the nuclear supply of cargo. On the basis of the crystal structure of PCI, a hydrophobic cavity has been characterized as the binding site for PE, which is recognized by specific lipids of conical morphology. Our findings indicate cell surface PE as a mediator for the cell membrane translocation of proteins and suggest that this requires the ability of the lipid to foster formation of transient nonbilayer domains within the membrane.
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Results |
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To quantify the cellular internalization of PCI, neutrophils were first exposed to [125I]PCI followed by the addition of a ligand dissociation buffer to exhaustively remove any proteins bound to the extracellular surface (Liu et al., 2000). Within the first 2 min of incubation at 37°C, a large proportion of PCI accumulated within the cells (Fig. 3 A).
The incorporation then proceeded at a slower rate. The quantity of PCI internalized at 6°C amounted to 2/3 of the uptake at 37°C (Fig. 3 A). In contrast to the PCI internalization, the PE transferase activity of PCI was clearly diminished at low temperatures. Indeed, PCI failed to increase the [14C]PE/[3H]PC ratio at 6°C (Fig. 3 B). To further address the participation of endocytic routes, CHO cells were transfected with the K44A mutant of dynamin2 (Altschuler et al., 1998), which prevents the budding of vesicles necessary for internalization of cargo via several major endocytic routes. Intrusion of Alexa 488–PCI into K44A-transfected cells was only slightly lowered compared with the uptake observed in the wild-type cells (Fig. 3 C). Also, transfection with the AP180 variant AP180-C selectively repressing the clathrin-mediated pathway barely reduced PCI incorporation (unpublished data). A major proportion of PCI incorporation thus persists under conditions suppressing the classic endocytic pathways. Inclusion of heparin into the cell suspensions to prevent interaction of PCI with the cell surface (Priglinger et al., 1994) substantially lowered the cell entry of PCI (Fig. 3 D). In contrast, the internalization was largely unaffected by receptor-associated protein, which blocks the function of the low-density lipoprotein receptor–related protein (LRP/
2 macroglobulin receptor), a mediator of the uptake of Tat (Liu et al., 2000). Heparin also abrogated the PCI-mediated cellular import of [14C]PE, as is evident from its ability to prevent the PCI-elicited increase of the [14C]PE/[3H]PC ratio (Fig. 3 E).
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Membrane insertion of PCI is facilitated by cone-type lipids
Because several of the translocating peptides were previously shown to generate transmembrane pores (magainin-2 and mastoparan X; Matsuzaki et al., 1995; Schwarz and Arbuzova, 1995), we investigated whether PCI elicited pore formation. As a positive control, the peptide mastoparan X rapidly evoked nucleation of pores in large unilamellar vesicles (LUV; Fig. 5 A), as was evident from the leakage of intravesicular carboxyfluorescein (CF).
In contrast, PCI did not elicit the formation of leakage sites for CF (Fig. 5 A), even after prolonged incubation (up to 120 min; not depicted). To evaluate whether PCI altered the membrane barrier properties for a small hydrophilic molecule, we monitored the membrane passage of the cation praseodymium (Pr3+; Sillerud and Barnett, 1982). By using 31P nuclear magnetic resonance (NMR), two peaks originating from the phosphate groups of the outer and inner monolayer phospholipids could be discerned (Fig. 5 B). In LUV containing solely PC, the peak pattern was unchanged by prolonged incubation with the cation. However, the addition of PCI markedly reduced the height and broadened the peak representing the inner leaflet phospholipids when long chain PE was present (Fig. 5 B). This demonstrates a direct interaction of Pr3+ with the inner monolayer phospholipids. As a negative control, no changes in the inner monolayer peak were seen without PCI. PCI thus generates transient and subtle changes in the bilayer structure that result in the formation of an aqueous pathway within the membrane.
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Partitioning of PCI into the lipid bilayer could be facilitated by a specific binding to PE (e.g., via interactions with the ethanolamine head group) and/or changes in the physicochemical properties of the membrane. In the latter case, nonbilayer domains, which are particularly favored by the conical shape of PE, could be involved (Israelachvili, 1980; de Kruijff, 1997). When the LUV were prepared from a short chain species of PE with a cylindrical morphology (di-14:0-PE), the protein was entirely degraded by extravesicular trypsin, similar to the findings obtained with the pure PC vesicles (Fig. 5 D). However, in LUV consisting of a lipid with a conical configuration but devoid of the ethanolamine head group DAG, PCI was partially resistant toward cleavage by the protease (Fig. 5 D). This suggests that the conical shape of long chain PE is essential for the membrane penetration of PCI. To further substantiate the extent of membrane insertion of the serpin, we exploited the ability of proteinase K to excessively degrade the serpin (Fig. 5 E). When the protease was integrated into the aqueous core of vesicles containing long chain PE, PCI was cleaved into several high molecular mass degradation products (Fig. 5 E), confirming that PCI protrudes into the vesicle core. In LUV supplemented with DAG, very similar fragmentation products were apparent. Conversely, after inclusion of the protease into vesicles only consisting of PC, the integrity of the protein was fully conserved (Fig. 4 E). This confirms that the serpin failed to reach the vesicle core in the presence of PC alone. Overall, our findings suggest that the conical shape of long chain PE is essential for the membrane penetration of PCI, which adopts a location that completely spans the bilayer.
PCI translocation requires key components of the hydrophobic pocket allowing selectivity of lipid interaction
Molecular docking studies based on the crystallographic structure of PCI (Huntington et al., 2003) were undertaken to characterize the structural components of PCI enabling its distinctive interaction with phospholipids. In view of the flexibility of the entire PE molecule, we chose a rigid fragment of the saturated hydrocarbon chain (C11 fragment) for the initial docking analysis because saturated fatty acyl chains are esterified to the C1 atom of the glycerol backbone of (diacyl-)PE under biological conditions (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200707165/DC1). Docking of the C11 fragment onto the PCI structure revealed 276 clusters with energies (kJ/mol) better than –15 among a total of 25,242,070 calculations. The two best clusters were nearly identical in position and energy (both better than –28 kJ/mol) and were significantly separated from the other clusters, with the next best hit at –24.3 kJ/mol. The best-docked C11 fragment fit into a capped hydrophobic pocket along helix D (D' channel; Fig. 6 A).
The orientation of the saturated chain was thus fixed. Consequently, the possible positions of the unsaturated chain, canonically esterified to the C2 atom of PE, were spatially limited to the same region. Of the top 10 clusters, two provided a portion of the possible binding site for the unsaturated hydrocarbon chain (9th and 10th best energies). It was found to be best accommodated by a second hydrophobic channel running along helix H (H' channel), in close proximity to the PCI-binding site for heparin (Figs. 6 A and S4, helix H). Moreover, in view of the locations revealed for the acyl chains, the position of the head group of PE was also suggested. The entire PE molecule was built on the two hydrocarbon chains placed by the docking studies and subjected to energy minimization. In the final model (Fig. 6 A), the ethanolamine group is found to be located in close proximity to Leu78, whereby the formation of an H bond between the N atom of the ethanolamine group and the main chain oxygen of Leu78 could be enabled. Space limitations within this region principally impair the interaction of PCI with phospholipids containing larger head groups than PE such as PC.
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Discussion |
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PE is the eminent plasma membrane lipid with a cone-type morphology because the concentrations of similarly shaped lipids (DAG and phosphatidic acid) are considerably lower. Mechanistically, DAG also efficiently supported the membrane penetration of PCI, whereas the protein intrusion was prevented by cylindrical PE species. This suggests that the conical shape of PE is of greater relevance for the interaction with PCI than specific interactions with the ethanolamine head group. Despite the bilayer-spanning location of PCI, the protein was not entirely transferred to the vesicle core, suggesting that its incorporation into the cell interior requires additional membrane components. Moreover, although transmembrane pores were not implicated in the PCI translocation, which is consistent with the inability of the conical lipids to support the pore formation (Matsuzaki et al., 1998), the serpin was found to elicit subtle perturbations of the membrane organization, as indicated by the leakage of ions from the lipid vesicles. This indicates that the PCI–PE interaction induces the formation of aqueous compartments within the membrane. Lipids of cone-type morphology are principally capable of generating areas of negative curvature in membranes (Farsad and De Camilli, 2003). This can facilitate the formation of intramembrane nonbilayer domains (inverted micelles), which might assist in the formation of aqueous compartments within the membrane. At present, it is unclear how the PCI–PE interaction mediated by the docking of the lipid to a distinct hydrophobic groove in PCI in the molecular system (see last paragraph of Discussion) relates to the alterations in physicochemical properties by the cone-shaped lipids that allow PCI transfer across the membrane. Hence, more in-depth analyses will be required in future studies to clarify whether these two observations are indeed interrelated. Overall, our results indicate that the membrane passage of PCI is crucially driven by lipids of a distinct morphology that generate an aqueous translocation route for the protein via the generation of nonbilayer domains.
The lipid-dependent internalization of cell-penetrating PCI principally agrees with a model theoretically proposed for the internalization of the translocating peptides (Joliot and Prochiantz, 2004). However, a contribution of PE and its peculiar shape for the cellular uptake of proteins has never been revealed. Cone-shaped lipids faciliate membrane fusion and budding processes (McMahon and Gallop, 2005), which are required for the formation of phagosomes. Consequently, the enhancement of bacterial phagocytosis induced by the PCI–PE interaction as observed here could be enabled by the stimulation of phagosome maturation. The ability of PCI to foster removal of pathogens suggests its participation in host defense and thus overlaps with the functions of other cell-penetrating proteins implicated in innate immunity. The internalized PCI was found to be rapidly directed to the nucleus, both in vitro and in the intact organism. Our findings position PCI as a promising candidate for the rapid delivery of cargo to the nucleus, including viruses, nucleotides of differing length and structures, and proteins/peptides. Being a component of the human proteome, potential applications of cargo-loaded PCI in humans are not immunogenic. PCI might be exploited in particular for the targeting of cargo into cells with increased surface exposure of PE (Vance, 2003), including capacitated sperm (Gadella and Harrison, 2000) and others.
PCI is shown to selectively extract PE from lipid particles, consecutively inserting the lipid into the plasma membrane of various cells. PCI thus belongs to a new class of proteins promoting the cellular uptake of selective lipids, including those specific for phosphatidylinositol (Wang and Munford, 1999) and sphingoymyelin (Stoeckelhuber et al., 2000). The latter proteins do not share structural homologies with PCI, a situation known from the intracellular lipid transfer proteins (Wirtz, 1997). During the transfer, the phospholipid would have to be largely protected from the contact with water, as revealed for scavenger receptor class B type I, the prototype mediator of the cellular phospholipid import (Urban et al., 2000). Based on the velocity, extent, and temperature dependence of the PE import, we assume that a principally similar process allows PCI to ferry PE into the cells. Only a limited change in the conformation of the protein–lipid complex might be required to switch between the extra- and intramembrane modes of the PCI–PE interaction. Indeed, the protein is most likely integrated into aqueous compartments during the membrane translocation. Nonetheless, at low temperatures, the lipid transferase activity of PCI is suppressed, whereas the PE-dependent PCI internalization is largely maintained. This indicates that the PE transfer is energy independent, whereas the membrane translocation of PCI exhibits a limited energy requirement, a property common to several cell-penetrating proteins. Whereas PCI facilitates PE enrichment on the cell surface by virtue of its lipid transferase activity, PE accumulation is further assisted by the delayed transbilayer movement of the lipid (Morrot et al., 1989). We observe that PE insertion accelerates the activity of the prothrombinase complex, thereby amplifying the formation of thrombin, a major coordinator of blood coagulation. Once internalized, PCI could potentially also support the intracellular movement of PE, such as between the cell membrane and membranes of intracellular organelles. Overall, our findings characterize PCI as the first transferase specifically fostering the cell import of PE.
Using the crystal structure of PCI as a template, we revealed that the selective interaction of PCI with PE is facilitated by a dual-channelled hydrophobic groove that is generated by the shortened A helix and a truncated loop between strands 3 and 4B. The H' and D' channels arising therefrom accommodate the extensions of the fatty acyl chains of the phospholipid with sharp precision. Indeed, because the H' channel is not capped, it can house hydrocarbon chains of up to 22 C atoms, the longest unsaturated fatty acids esterified to PE in mammals. Moreover, the restricted size of the structure in which the phospholipid headgroup is buried markedly favors interactions with conical phospholipids. Hydrogen bonds between the ethanolamine group and specific amino acids, suggested to permit the folding of bacterial lactose permease (Bogdanov et al., 1999), might additionally ease the interaction with the phospholipid, as exemplified by the functional contribution of Leu78. Together, this structural constellation is supposed to enable the higher affinity of PCI for PE over PC. The docking and energy minimization studies were corroborated by the mutagenesis of single amino acids designed to specifically remodel the hydrophobic cavity. In particular, the insertion of a triple Ala sequence on top of the H' channel substantially promoted the intrusion and membrane translocation of the serpin. The insertion, which reconstitutes the truncated loop intercalated between strands 3 and 4B, is likely to strengthen the interaction with the fatty acyl chains of the phospholipids by virtue of its specific location and enhanced hydrophobicity. In conclusion, the hydrophobic pocket appears to represent a unique structure for accommodating lipids capable of supporting the membrane passage of proteins.
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Materials and methods |
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Preparation of native PCI and PCI variants
For preparation of recombinant PCI, the cDNA of human PCI was amplified from a human liver cDNA library using a forward (5'-CCGCTCGAGCATATGCACCGCCACCACCCCCGGGAG-3') and reversal primer (5'-CGGGATCCATCATCAGGGGCGGTTCACTTTGCCAAG-3'). Mutagenesis was performed using the QuickChange site-directed mutagenesis kit (Stratagene). The following mutants were generated: V374W, T341R, L78W, R35A, and 376AAA. All mutations were confirmed by DNA sequencing. After transformation of E. coli with the plasmids, 0.4 mM IPTG was added to induce the expression of the proteins. Bacterial supernatants were applied to a TALON Metal Affinity Resin column (Clontech Laboratories, Inc.) and the fractions eluted from the column were analyzed by SDS-PAGE. Urinary PCI (uPCI) was purified as described previously (Priglinger et al., 1994).
Phospholipid exchange
For the determination of the intervesicular phospholipid exchange, py-labeled donor vesicles and unlabeled acceptor vesicles were coincubated for different time periods, the suspensions were passed over an anion exchange column (Bio-Rad Laboratories), and the fluorescence was determined in Triton X-100 (Sigma-Aldrich) solubilisates of the acceptor vesicles as described previously (Stoeckelhuber et al., 2000). The vesicle preparations and incubations were performed in the presence of 10 nM butylated hydroxytoluene and under argon or nitrogen. The transfer of py-labeled phospholipids into the cells was analyzed as described previously (Urban et al., 2000). Under principally similar conditions, the cells were incubated with lipid donor vesicles containing traces of [14C]PE (1-palmitoyl, 2-linoleoyl species) and [3H]cholesterol (both from GE Healthcare).
Confocal microscopy
Washed HL-60 cells and murine leukocytes (see Incorporation of PCI...) were incubated with biotin-labeled PCI at 6 or 37°C. The cells were fixed with 4% paraformaldehyde for 60 min at room temperature, permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min at 4°C, and washed thereafter. Nonspecific binding sites were saturated overnight at 4°C with antibody dilution buffer (Dako) containing 0.2% goat serum. Subsequently, the cells were incubated for 60 min with Cy-2–labeled streptavidin (GE Healthcare) and an additional 30 min with TO-PRO-3 (Invitrogen). The cells were transferred onto slides and covered with Vectashield (Vector Laboratories). Slides were analyzed at room temperature with a laser scanning microscope (LSM 510 Meta; Carl Zeiss, Inc.) using a Plan-neofluar 40x, 1.3 oil differential interference contrast 1056–602 (1083–997) objective lens (Carl Zeiss, Inc.) and 488-nm Ar2 and 633-nm HeNe2 lasers. Images were obtained with a PMT photomultiplier tube (Hamamatsu Photonics). Image acquisition and processing was performed with LSM 510 image examiner software (Carl Zeiss, Inc.).
For analysis of phagocytosis of E. coli into THP-1 macrophages, the cells (105/well) were seeded in 24-well plates in 500 µl RPMI-1640 supplemented with 10% heat-inactivated FCS, 1% Hepes, 500 U/ml penicillin, and 500 µg/ml streptomycin and differentiated by the addition of 10 nM PMA. After 16 h, the cells were fed with fresh, PMA-free medium and cultivated for two more days. The macrophages were incubated with Alexa 488–labeled uPCI or FITC E. coli alone or together with their nonfluorescent counterparts in serum-free medium. Some samples were treated with duramycin (Sigma-Aldrich) for 15 min before the start of the incubations. The samples were visualized with a confocal laser scanning microscope (LSM 510 META). The images were taken by using a 63x Plan-Apochromat oil objective (Carl Zeiss, Inc.). Alexa 488 and FITC were irradiated at 488 nm and detected via a 505 longpass filter (Carl Zeiss, Inc.). The images obtained were analyzed and quantified using LSM Image Browser software (Carl Zeiss, Inc.).
Immuno EM
HL-60 cells previously incubated with or without 90 nM of biotin-labeled PCI were washed and fixed with 4% paraformaldehyde/5% glutaraldehyde for 60 min at room temperature. Before embedding in LR white medium resin (Pelco), the HL-60 cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer and then rinsed in 0.1 M phosphate buffer. Ultrathin serial sections of 150 nm were cut and mounted on gold grids. Nonspecific binding sites on the sections were blocked with 5% BSA in PBS, and grids were incubated for 120 min at 37°C with 10 nm of gold-labeled goat anti-biotin IgG (Biocell Laboratories, Inc.). The sections were then counterstained with 3% uranyl acetate and lead citrate and examined with a transmission electron microscope (JEM TEM 1200 EXII; JEOL Ltd.).
Incorporation of PCI by murine leukocytes in vivo
4 µg of biotin-labeled recombinant PCI (rPCI) was injected into the tail vein of anaesthetized mice. After 30 min, blood was drawn from the periorbital sinus into 0.38% sodium citrate, immediately diluted by 1:10 with erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA, pH 7.4), and centrifuged at 400 g for 20 min. The pellets were washed three times with decreasing volumes (1.5, 0.5, and 0.1 ml) of erythrocyte lysis buffer. The leukocytes obtained were processed for confocal microscopy as described previously.
Phagocytosis assay
THP-1 cells were differentiated with PMA as described in Confocal microscopy. Subsequently, the cells were washed in serum-free medium, mixed with 2 x 107 FITC-labeled E. coli (Vybrant Phagocytosis Assay kit; Invitrogen) in a final volume of 500 µl, and incubated for 2 h at 37°C in the presence or absence of 100 nM uPCI, 50 µM cytochalasin D, or 10% (vol/vol) human serum. In the preincubation experiments, either macrophages or bacteria were separately preincubated with 100 nM uPCI in serum-free medium for 60 min at 37°C and washed once before being added to the phagocytosis assay. After 2 h, noninternalized bacteria were rinsed away (after rinsing fluoroscence images were taken) and loosely attached E. coli were removed by trypsinization. The phagocytes were collected by centrifugation, resuspended in ice-cold PBS, and analyzed by flow cytometry (FACSCalibur; BD Biosciences).
Quantification of cellular internalization of PCI
uPCI was labeled with 125I (GE Healthcare) by adding 100 µCi of the isotope to 50 µl of 0.25 M sodium phosphate buffer, pH 7.0, followed by the immediate mixing with 10 µl of 5 mg/ml chloramine T solution. 5 min after addition of 2.5 µg PCI, the reaction was stopped by inclusion of 50 µl of a saturated sodium ascorbate buffer, pH 7.0, and the labeled protein was purified from the unreacted 125I via passage over a Sephadex-G10 column (GE Healthcare). After incubation with the labeled PCI, the cells were washed once with PBS and incubated for 30 min at 4°C with ligand- dissociation buffer (0.05% trypsin, 0.5 M EDTA, and 50 µg/ml proteinase K in PBS; Liu et al., 2000). The suspensions were centrifuged, the extracellular media were discarded, and the radioactivity of the pellets was determined. Alexa fluorescence intensity was measured in detergent-treated pellets (2% Triton X-100).
Prothrombinase activity
The activity of the prothrombinase complex was determined as described previously (Zieseniss et al., 2001).
Western blots
HL-60 cells were incubated with 100 nM PCI at 37°C, and the cell pellet was resuspended in nuclear extraction buffer 1 (10 mM Tris-HCl, 10 mM KCl, 0.5% IGEPAL CA-630, and protease inhibitor cocktail Complete; Roche), incubated for 30 min on ice, and lysed by sonication. The supernatants obtained from the homogenates were centrifuged and the resulting supernatants were used for separation of the cytosolic proteins. The pellets of the homogenates containing the nuclei were resuspended in extraction buffer 2 (10 mM Tris-HCl, 0.5M NaCl, and Complete), incubated for 30 min on ice, and centrifuged for 30 min, and the supernatants were recovered. After 10% SDS-PAGE, the proteins were transferred onto polyvinylidene fluoride membranes (Millipore), blocked overnight with 5% dry milk, and incubated for 60 min with anti-PCI antibody. After incubation for 45 min with horseradish peroxidase–conjugated anti–rabbit IgG, the proteins were visualized.
Translocation of PCI across pure lipid membranes
LUV were prepared from 10 mg of total lipids per milliliter in a buffer composed of 20 mM Hepes and 150 mM NaCl, pH 7.5, and supplemented with 0.5 mg/ml trypsin or 0.1 mg/ml proteinase K (intravesicular proteases). The various lipids (1-palmitoyl, 2-linoleoyl species of PC and PE, dimyristoyl-PE, dioleoyl-DAG, and PS) were dispersed by vortexing with glass beads. They were passed several times through an extruder (100 or 200 nm filters; LiposoFast; Avestin) until the solution was clear. The vesicle suspensions were subsequently dialyzed. Then the vesicle suspensions were incubated with PCI for 60 min at 37°C. The vesicle preparations and incubations were performed in the presence of 10 nM butylated hydroxytoluene under argon or nitrogen. Incubations with the intravesicular proteases were performed in the presence of 2 mM of the protease inhibitor PMSF. Where indicated, the preparation of the LUV was performed before the proteases, and trypsin or proteinase K were added after the incubations with PCI (extravesicular proteases).
Efflux of CF from LUV
The lipids were dispersed in a buffer composed of 10 mM Hepes, 100 mM KCl, and 1 mM EDTA, pH 7.4, containing 60 mM CF (mixed isomers; Sigma-Aldrich). The vesicles were sized using 200-nm polycarbonate filters and separated from the extravesicular CF by dialysis. The fluorescence signal F was recorded after addition of mastoparan X and PCI at 37°C. In the case of pore formation, dequenching of the dye upon dilution into the medium will increase the values above the initial value Fo. To determine the fluorescence signal for the maximally releasable amount of CF (Ft), the vesicles were solubilized using the detergent C12E8.
Fluorescence correlation spectroscopy
Giant unilamellar vesicles (GUV) were prepared from 1-palmitoyl, 2-linoleoyl species of PC and PE together with 5 mol% dipalmitoyl-PS. For determination of the translational diffusion coefficient DT, 10–4 mol% of DiD-C18 (Invitrogen) was added. The dispersed lipids were transferred to a coverglass chamber (Lab-Tek; Nalge Nunc), the formed GUV were allowed to settle for 20 min, and 9 nM Alexa 488–PCI was added. The fluorescence correlation spectroscopy measurements were performed in the autocorrelation mode with the ConfoCor 2 system (Carl Zeiss, Inc.) using either the 488-nm line of an Ar-ion Laser or a 633-nm HeNe Laser (at 50–60 µW). The excitation light was reflected by a dichroic mirror (HFT 488/633; Carl Zeiss, Inc.) and focused onto the sample. The emitted fluorescence light was split by a second dichroic mirror (NFT 635; Carl Zeiss, Inc.), passed through two filters (505–550 nm bandpass and 650 nm longpass filter; Carl Zeiss, Inc.), and detected in two separate channels. In each case, out of plane fluorescence was reduced by a pinhole of 90-µm diameter. For analysis of the membrane interaction of PCI, the fluorescence correlation spectroscopy focal spot was positioned near the center of the upper membrane of the GUV. For characterizing the diffusion properties of PCI in aqueous solution, the focus was moved to a position 20 µm above the upper membrane. The data were evaluated by Levenberg-Marquardt nonlinear least squares fitting to the appropriate model of the autocorrelation function.
Pr3+ transfer into vesicles
LUV of a diameter of 200 nm were prepared by the extrusion method (see Translocation of PCI...) and Pr3+ was added to a final concentration of 5.7 mM. The chemical shifts of the 31P signals from the phospholipids present in the outer and inner leaflet of the LUV were registered. NMR spectra were recorded with a spectrometer (400S; Varian Medical Systems).
Docking studies
Based on the crystal structure of cleaved PCI (Huntington et al., 2003), docking studies for PE binding were performed with the program DockVision. Because of the extreme flexibility of the PE ligand, the docking studies were initially performed on a straight 11-carbon chain fragment (generated using CORINA; Huntington et al., 2003) meant to represent a possible conformation of the fully saturated hydrocarbon chain of PE. The maximum floating radius was set at 100 Å to ensure full coverage of PCI, and a total of 10,000 trials with a schedule of six conditions of 500 steps each was used in docking. The model of the PE–PCI complex using the 1-palmitoyl, 2-linoleoyl species of PE was built in XtalView (Huntington et al., 2003) and energy minimized using CNS (one cycle of 2,000 steps; Brunger et al., 1998).
Statistics
The mean values given are ± SD. The determinations were compared by one-way analysis of variance. Differences of P < 0.05 were considered to be significant.
Online supplemental material
Fig. S1 shows that inhibition of PCI decreases PE import into activated platelets. Fig. S2 indicates that uPCI added to HL-60 cells is incorporated into nuclear fractions of those cells. Fig. S3 shows that the PE-dependent internalization of PCI is also detected at low temperatures and under conditions of extensive proteolysis of cell surface proteins. Fig. S4 provides results from molecular docking analyses indicating the presumed PE binding site in PCI. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200707165/DC1.
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Acknowledgments |
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This study was supported by grants from the Deutsche Forschungsgemeinschaft to B. Engelmann and from the Austrian Science Foundation (projects P16093-B04 and P17337-B09) to M. Geiger.
Submitted: 24 July 2007
Accepted: 24 October 2007
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References |
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). Similar findings were obtained with rPCI (not depicted). (C) PCI selectively enhances the cellular import of PE. Isolated human platelets were incubated at 37°C for 10 min with uPCI and SUV containing the indicated py-phospholipids. Gray, control; black, PCI. n = 3–6. *, P < 0.05. (D) PCI supports cell uptake of [14C]PE. Platelet associated radioactivity was determined after incubation with SUV (95:5% PC/PE; 14C-PE and 3H-PC) without (control) or with uPCI (10 min, 37°C). n = 5. *, P < 0.05 versus vesicle and control. (E) Facilitation of PE transfer increases thrombin formation. Platelets were challenged with 0.5 U/ml thrombin, a relatively weak stimulator of the prothrombinase activity, which nevertheless induces a robust secretion of PCI. In the presence of SUV containing PC alone (PC) or 5% PE plus 95% PC (PE; mol%), thrombin generation by the prothrombinase complex was evaluated with or without uPCI. Control experiments verified that no prothrombinase activity was elicited by the PE vesicles in the absence of platelets. An isotype control antibody did not inhibit the prothrombinase activity of activated platelets in the presence of PC/PE vesicles (not depicted). n = 4–6. *, P < 0.05. Results show mean values ± SD.
