Poly-N-acetyllactosamine structures on murine cell surface T200 glycoprotein participate in natural killer cell binding to YAC-1 targets

Cell-surface murine T200 glycoprotein has been implicated in the binding of NK cells to certain susceptible tumor targets. The existence of poly-N-acetyllactosamine structures on T200 glycoprotein and the ability of lactosamine-type oligosaccharides to inhibit NK cell-mediated cytotoxicity suggest that these structures may also be important in NK-target binding. To further identify and characterize these structures, relevant saccharides and reconstituted membrane liposomes containing fractionated effector cell membrane proteins were tested for their ability to block conjugate formation. Under base line conditions, the majority of plastic-non-adherent, Percoll-fractionated, NK-enriched splenocytes that formed conjugates with NK-susceptible YAC-1 targets functioned as lytic effectors in a single-cell cytotoxicity assay. These effectors were blocked in their ability to bind to YAC-1 targets by the addition of N-acetyllactosamine [Gal(beta 1,4)-GlcNAc] and chitobiose [GlcNAc(beta 1,4)GlcNAc], but not by saccharides lacking lactosamine-type linkages. Liposomes prepared from octyl-beta-D-glucopyranoside-extracted YAC-1 and NK-enriched effector cell membranes interfered with conjugate formation, whereas liposomes prepared from NK-insensitive P815 cells were inconsequential. Surface radiolabeled effector cell membrane proteins were fractionated by tomato lectin-Sepharose 4B (poly-N-acetyllactosamine-specific) column chromatography. Tomato lectin-bound material was enriched in a glycoprotein identical with T200, which, when incorporated into liposomes, was a potent inhibitor of effector-target binding. This inhibitory capacity was abrogated by treatment of liposomes with Ly-5 mAb (T200 mAb) or the lactosamine-specific enzyme endo-beta-galactosidase. When T200 was purified by mAb affinity chromatography and incorporated into liposomes, it was a potent inhibitor of conjugate formation, an effect that was blocked by pretreatment of T200-containing liposomes with Ly-5 mAb or endo-beta-galactosidase. These data provide additional evidence that T200 can mediate binding of NK cells to YAC-1 targets, and that poly-N-acetyllactosamine-type structures on NK cell surface T200 glycoprotein are important in the binding process.     

A sensitive method to introduce membrane-bound proteins into recipient cells based on affinity enrichment of lipid vesicles to the recipient cell prior to fusion

A sensitive analytical procedure for studying membrane-bound structures has been developed. Membrane glycoproteins inserted into liposomes were transferred to recipient cells by use of a lectin, concanavalin A, bound to the cells as a bridge to generate proximity between the recipient cell and the glycoprotein-containing liposome, prior to exposure to the fusing agent, poly(ethylene glycol). Partially purified histocompatibility antigen from rats was introduced into the membrane of human lymphocytes. After treating the cells with poly(ethylene glycol) under fusion conditions, some of the antigen present in the preparation could not be eluted with alpha-methyl mannoside and EDTA, indicating that incorporation in the cell membrane had taken place. This antigen remained exposed on the lymphocyte surface for approximately 1 h as demonstrated by sensitivity of the lymphocytes to the lytic effect of an antiserum to the histocompatibility antigen in the presence of complement. Some of the lectin molecules seemed to be internalized in the cells but no induction of cell mitosis was observed. The described method gives an opportunity to work with small amounts of membrane proteins inserted into liposomes, introducing them into recipient cells for analysis of their biological activities.     

Fluorescent lipid probes in the study of viral membrane fusion

Fluorescent lipid probes are widely used in the observation of viral membrane fusion, providing a sensitive method to study fusion mechanism(s). Due to the wealth of data concerning liposome fusion, a variety of fusion assays has been designed including fluorescent probe redistribution, fluorescence dequenching, fluorescence resonance energy transfer and photosensitized labeling. These methods can be tailored for different virus fusion assays. For instance, virions can be loaded with membrane dye which dequenches at the moment of membrane merger. This allows for continuous observation of fusion and therefore kinetic information can be acquired. In the case of cells expressing viral envelope proteins, dye redistribution studies of lipidic and water-soluble fluorophores yield information about fusion intermediates. Lipid probes can be metabolically incorporated into cell membranes, allowing observation of membrane fusion in vitro with minimal chance of flip flop, non-specific transfer and formation of microcrystals. Fluorescent lipid probes have been incorporated into liposomes and/or reconstituted viral envelopes, which provide a well-defined membrane environment for fusion to occur. Interactions of the viral fusion machinery with the membrane can be observed through the photosensitized labeling of the interacting segments of envelope proteins with a hydrophobic probe. Thus, fluorescent lipid probes provide a broad repertoire of fusion assays and powerful tools to produce precise, quantitative data in real time required for the elucidation of the complex process of viral fusion.     

"Smart" drug carriers: PEGylated TATp-modified pH-sensitive liposomes

To engineer drug carriers capable of spontaneous accumulation in tumors and ischemic areas via the enhanced permeability and retention (EPR) effect and further penetration and drug delivery inside tumor or ischemic cells via the action of the cell-penetrating peptide (CPP), we have prepared liposomes simultaneously bearing on their surface CPP (TAT peptide, TATp) moieties and protective PEG chains. PEG chains were incorporated into the liposome membrane via the PEG-attached phosphatidylethanolamine (PE) residue with PEG and PE being conjugated with the lowered pH-degradable hydrazone bond (PEG-HZ-PE). Under normal conditions, liposome-grafted PEG "shielded" liposome-attached TATp moieties since the PEG spacer for TATp attachment (PEG(1000)) was shorter than protective PEG(2000). PEGylated liposomes are expected to accumulate in targets via the EPR effect, but inside the "acidified" tumor or ischemic tissues lose their PEG coating due to the lowered pH-induced hydrolysis of HZ and penetrate inside cells via the now-exposed TATp moieties. This concept is shown here to work in cell cultures in vitro as well as in ischemic cardiac tissues in the Langendorff perfused rat heart model and in tumors in experimental mice in vivo.     

Defining cytolytic T lymphocyte recognition of chemically modified self. I. Response to trinitrophenyl-H-2Kk

We used purified class I antigen incorporated into liposomes to examine the response of secondary cytolytic T lymphocytes (CTL) to chemically modified self. By generating the secondary response in the presence of T cell helper factor, the level of CTL response was limited by CTL recognition of added antigen rather than by helper cell generation of lymphokines. We found a strong secondary response against chemically modified self when spleen cells from trinitrophenyl (TNP)-primed C3H/HeJ mice were stimulated with a) TNP-modified liposomes containing H-2Kk, b) liposomes containing H-2Kk purified from TNP-modified RDM-4 (H-2k) cells, or c) liposomes containing the limited trypsin proteolysis product of H-2Kk that had been directly modified with TNP. In contrast, we were not able to generate a significant CTL response with unmodified H-2Kk incorporated into vesicles along with TNP-modified membrane components lacking H-2Kk. These results suggest that TNP-modified H-2Kk is a major antigenic site recognized by CTL from C3H/HeJ mice after priming against TNP-modified self.     

Cell-mediated immunity to herpes simplex virus: induction of cytotoxic T lymphocyte responses by viral antigens incorporated into liposomes

The immunogenicity of inactivated herpes simplex virus type 1 (HSV-1) antigens incorporated into liposomes was measured by their ability to induce secondary anti-HSV-1 specific cytotoxic T lymphocyte (CTL) responses in splenocyte cultures from virus-primed mice. Such virus-specific CTL could be induced provided the liposomes contained virus along with plasma membrane antigen of the same H-2 type as that of the virus-primed responser cells. Responses did not occur in cultures stimulated with liposomes containing only viral antigens or with a mixture of liposomes composed respectively of lipid and virus with those composed of lipid and plasma membrane proteins. Moreover F1 responder cells stimulated with liposomes composed of virus and plasma membrane protein of one of the parental haplotypes produced CTL restricted in their cytotoxicity to infected targets of the same haplotype as was used in the liposome. These results show that liposomes can be used to induce anti-HSV-1 CTL with inactivated viral antigens but recognition of both viral and H-2 antigen is required for this process to occur in vitro. The implications of our findings to the preparation of subunit vaccines against HSV-1 are briefly discussed.     

The role of target membrane sialic acid residues in the fusion activity of the influenza virus: the effect of two types of ganglioside on the kinetics of membrane merging

The influenza virus enters target cells via the action of hemagglutinin proteins (HA) inserted into the viral envelope. HA promotes membrane fusion between the viral envelope and endosomal membrane at low pH, following viral binding to sialic acid-containing receptors on target cells, and internalization by endocytosis. The effect of target membrane sialic acid residues on the fusion activity of the influenza virus towards model membranes was evaluated by both reduction, (i.e. treating somatic cells with neuraminidase- (NA-) prior to virus-cell interactions), and by supplementing liposomes with the gangliosides GD1a and GT1b. The harshness of the neuraminidase pretreatment of target cells required to affect virus-induced membrane merging was found to greatly depend on the assay conditions, i.e. whether a virus-cell prebinding step at neutral pH was included prior to acidification. Minor concentrations of neuraminidase were found to greatly reduce virus fusion, but only in the absence of a prebinding step; they had no effect if this step was included. Although membrane merging was greatly reduced following cell neuraminidase pretreatment, virus-cell association at low pH was not disturbed proportionately. This probably reflects unspecific virus-cell binding under these conditions, probably of inactivated or aggregated virus particles, which does not translate into membrane merging. This seems to suggest both that target membrane sialic acid can protect the virus from losing its activity before triggering membrane merging, and that the importance of this interaction is not merely to ensure virus-target proximity. With liposomes, we found that both types of ganglioside supported efficient fusion, with GD1a promoting a slightly faster initial rate. However, in this case, virus-target proximity closely mirrored fusion activity, thus pointing to differential specificity between targets routinely used to assay influenza virus fusion activity.     

Delivery of liposome membrane-associated sterols through silastic membranes

The transport of sterols incorporated into the lecithin bilayer of small unilamellar liposomes through a model membrane was studied. A two-chamber diffusion cell containing liposomes with incorporated [4-¹⁴C]cholesterol or β-[4-¹⁴C]sitosterol in the donor chamber and liposomes with unlabeled cholesterol in the receiver chamber was used. The permeability coefficients of the sterols through silastic rubber membranes which served as a model membrane were measured. The permeability for cholesterol incorporated into liposomes in a phosphatidyl choline/cholesterol molar ratio of 1 : 1, produced by sonication for 1 h, and subsequent centrifugation at 100000 × g for 1 h, was 1.6 · 10^?8 cm sec^?1. Dilution of the liposome suspension did not change the permeability coefficient significantly. The permeability coefficient of sitosterol incorporated into liposomes was about 4-times smaller than that of cholesterol. These results suggest that the sterols were delivered to the silastic membrane by the intact liposomes and that free solute was not involved in the transport to the membrane to a significant degree. The large differences in the permeability coefficients between cholesterol and sitosterol indicate that an aqueous interfacial barrier was crossed by the sterol during the delivery to the membrane.     

Labelling of liposomes with intercalating perylene fluorescent dyes.

The high fluorescent potential and the exceptional photostability of lipophilic derivatives of perylene-3,4:9,10-bis(dicarboximides) are utilized for the fluorescence-labelling of liposomes. The preparation of the liposomes is effected by supersonic starting from a lipid mixture consisting of the matrix lipids soy lecithin, cholesterol, alpha-tocopherol and the perylene dyes. From a multitude of perylene derivatives investigated only those are optimally incorporated into the bilayer membrane of unilamellar liposomes which are substituted at both nitrogen atoms by one or two linear hydrocarbon groups. In order to attain an optimal fluorescent quantum yield, about 200 to 300 dye molecules can be incorporated per liposome. The liposomes thus obtained have a diameter of about 70 to 80 nm, are homogeneous and may be stored for more than seven months. Neither the fluorescent properties nor the stability of these liposomes are influenced by the additional incorporation of various ara C-derivatives and lipophilic anchor groups which subsequently enable the coupling of antibodies to the liposomes. As the water-insoluble perylene dyes are incorporated into the bilayer membrane, the aqueous inner volume of the liposomes remains available for a further utilization.     

Properties of exogenously added GPI-anchored proteins following their incorporation into cells.

Isolated glycosylphosphatidylinositol (GPI)-anchored proteins, when added to cells in vitro, incorporate into their surface membranes and, once incorporated, exert their native functions. Virtually any protein of interest, if expressed as a GPI-reanchored derivative, can be modified to acquire this capacity. Such transfer of proteins directly to cells, termed "protein engineering" or "painting" constitutes an alternative to conventional gene transfer for manipulating cell surface composition that has many potential applications. Previous studies with incorporated GPI-anchored proteins have focused almost entirely on their extracellular functions. In this study, biotinylated human erythrocyte (E(hu)) decay accelerating factor, E(hu) acetylcholinesterase, and GPI-reanchored murine B7-1 and B7-2 were used as GPI-anchored reporters to characterize their plasma membrane organization and cell signalling properties following addition to Hela or Chinese hamster ovary cells. For each reporter, three types of cell-association were documented; (1) nonphysiological attachment and/or incomplete insertion, (2) uncomplexed membrane integration, and (3) organization into TX-100-resistant microdomains. Transit from the first two compartments into the third, i.e., microdomains, progressed slowly, continuing even after 24 to 36 h and was associated with the acquisition of cell signalling capacity. All four reporters, incorporated in two different detergents, behaved similarly. When organized in microdomains, caveolin and other GPI proteins co-isolated with the incorporated reporter. These results have implications for protein engineering of cells in general, and in particular, for cells such as modified tumor cell immunogens administered to patients for therapeutic purposes.     

Binding of endostatin to phosphatidylserine-containing membranes and formation of amyloid-like fibers

Endostatin, the 20-kDa C-terminal NC1 domain of collagen XVIII, is an endogenous inhibitor of tumor angiogenesis and tumor growth. A major problem in reconciling the many reported in vitro effects of endostatin is the lack of a high-affinity receptor, and a search for the latter continues. In accordance with the above, the molecular mechanisms of action of endostatin remain elusive. We show here that endostatin binds to membranes containing acidic phospholipids, phosphatidylserine (PS) or phosphatidylglycerol (PG). More specifically, a red shift in the fluorescence emission of Trp of endostatin in the presence of liposomes containing these anionic lipids was evident, revealing the average environment of Trps to become less hydrophobic. This shift was not observed for phosphatidylcholine (PC) liposomes, demonstrating the acidic lipid to be required. Quenching by endostatin of the fluorescence of a pyrene-labeled phospholipid analogue in PS containing membranes was seen, while there was no effect for PC liposomes. Resonance energy transfer from the Trp residues of endostatin to a dansyl-labeled phospholipid further confirmed the association of endostatin with PS-containing membranes, whereas there was no binding to PC liposomes. Intriguingly, the association of endostatin with PS-containing liposomes triggered the formation of fibers, with Congo red staining producing green birefringence characteristic for amyloid. Lipid was incorporated into these fibers, as shown by staining when a trace amount (X = 0.02) of fluorescent phospholipid analogues was present in the liposomes. No fiber formation was seen when endostatin was added to liposomes composed of PC only. Because PS has been reported to be exposed in the outer surface of the plasma membrane of cancer cells and vascular endothelial cells, our results suggest that this lipid could represent a target for endostatin in the cancer cell surface and tumors, thus suggesting a novel mechanism of its action. More specifically, analogous to a number of other cytotoxic proteins interacting with negatively charged lipids, PS-triggered fiber formation by endostatin on the surface of cancer cells would impair the permeability barrier function of the plasma membrane, resulting in cell death.     

Enhanced Neutrophil Stimulation by Phorbol Ester Encapsulated in pH-Sensitive Liposomes

Abstract

Phorbol 12-myristate 13-acetate (PMA) and arachidonic acid (AA) are both hydrophobic stimulators for superoxide release by guinea pig neutrophils. However AA incorporated into liposomes is no longer an effective stimulator. In contrast, PMA incorporated into liposomes is more effective in neutrophil stimulation than free PMA. the ED₅₀ of superoxide release was 3.1 × 10^?8M, and 4.0 × 10^?10 M for free PMA and liposomes composed of egg phosphatidylethanolamine (PE) /AA/ PMA (molar ratio 7:2:1), respectively. PMA incorporated into PE/AA liposomes could also shorten the lag period of superoxide release in a concentration-dependent fashion. the enhanced stimulation activity of PMA in liposomes was correlated with the enhanced liposome uptake by neutrophils, probably via phagocytosis. Weak bases and a proton ionophore inhibited superoxide release by cells stimulated with either free or liposomal PMA. these results suggested that free PMA attached to cell membranes might be endocytosed and stimulate the superoxide-generating systems via an endocytic compartment(s). Since liposomes effectively deliver the contents into the compartments, liposomal PMA may thus be a potent stimulator for neutrophils. This hypothesis is further supported by the observation that pH-sensitive liposomes, which are active in the acidic endocytic compartments, are more effective carriers for PMA than the conventional pH-insensitive liposomes.     

Evidence that the transmembrane biogenesis of aquaporin 1 is cotranslational in intact mammalian cells

Most polytopic membrane proteins are believed to integrate into the membrane of the endoplasmic reticulum (ER) cotranslationally. However, recent studies with Xenopus oocytes and dog pancreatic microsomes have suggested that this is not the case for human aquaporin 1 (AQP1). These experiments indicate that membrane-spanning segments (MSSs) 2 and 4 of AQP1 do not integrate into the membrane cotranslationally so that this protein initially adopts a four MSS topology. A later maturation event involving a 180-degree rotation of MSS 3 from an N(lum)/C(cyt) to an N(cyt)/C(lum) orientation and the concomitant integration of MSSs 2 and 4 into the membrane results in the final six MSS topology. Here we examine the biogenesis of AQP1 in the human embryonic kidney cell line HEK-293T. To do this, we constructed an expression vector for a fusion protein consisting of the enhanced green fluorescent protein followed by an insertion site for AQP1 sequences and a C-terminal glycosylation tag. We then transiently transfected HEK-293T cells with this vector containing the AQP1 sequence truncated after each MSS. Glycosylation of the C-terminal tag was used to monitor its location relative to the ER lumen and consequently the membrane integration and orientation of successive MSSs. In contrast to previous studies our results indicate that AQP1 integrates into the ER membrane cotranslationally in intact HEK-293T cells.     

Liposome-mediated transfer of integral membrane glycoproteins into the plasma membrane of cultured cells

Cultured mouse 3T3 cells treated with phosphatidylserine or phosphatidylserine/phosphatidylcholine (3: 7 mole ratio) liposomes containing ortho- and paramyxovirus envelope glycoproteins become susceptible to killing by virus-specific cytotoxic T lymphocytes indicating that the liposome-derived glycoproteins have been inserted into the cellular plasma membrane. Cells incubated with liposomes of similar lipid composition containing viral antigens plus a dinitrophenylated lipid hapten were killed by both virus- and hapten-specific T lymphocytes indicating that both protein and lipid components are inserted into the plasma membrane. We consider that assimilation of liposome-derived antigens into the plasma membrane results from fusion of liposomes with the plasma membrane. Cells incubated with phosphatidylcholine liposomes containing lipid haptens and viral glycoproteins were not killed by cytotoxic lymphocytes indicating that liposomes of this composition do not fuse with the plasma membrane. Liposome-derived paramyxovirus glycoproteins inserted into the plasma membrane retain their functional activity as shown by their ability to induce cell fusion. These experiments demonstrate the feasibility of using liposomes as carriers for introducing integral membrane (glyco)proteins into the plasma membrane of cultured cells and establish a new approach for studying the role of individual (glyco)proteins in the expression of specific cell surface properties.     

C8-glycosphingolipids preferentially insert into tumor cell membranes and promote chemotherapeutic drug uptake

Insufficient drug delivery into tumor cells limits the therapeutic efficacy of chemotherapy. Co-delivery of liposome-encapsulated drug and synthetic short-chain glycosphingolipids (SC-GSLs) significantly improved drug bioavailability by enhancing intracellular drug uptake. Investigating the mechanisms underlying this SC-GSL-mediated drug uptake enhancement is the aim of this study. Fluorescence microscopy was used to visualize the cell membrane lipid transfer intracellular fate of fluorescently labeled C₆-NBD-GalCer incorporated in liposomes in tumor and non-tumor cells. Additionally click chemistry was applied to image and quantify native SC-GSLs in tumor and non-tumor cell membranes. SC-GSL-mediated flip-flop was investigated in model membranes to confirm membrane-incorporation of SC-GSL and its effect on membrane remodeling. SC-GSL enriched liposomes containing doxorubicin (Dox) were incubated at 4 °C and 37 °C and intracellular drug uptake was studied in comparison to standard liposomes and free Dox.SC-GSL transfer to the cell membrane was independent of liposomal uptake and the majority of the transferred lipid remained in the plasma membrane. The transfer of SC-GSL was tumor cell-specific and induced membrane rearrangement as evidenced by a transbilayer flip-flop of pyrene-SM. However, pore formation was measured, as leakage of hydrophilic fluorescent probes was not observed. Moreover, drug uptake appeared to be mediated by SC-GSLs. SC-GSLs enhanced the interaction of doxorubicin (Dox) with the outer leaflet of the plasma membrane of tumor cells at 4 °C. Our results demonstrate that SC-GSLs preferentially insert into tumor cell plasma membranes enhancing cell intrinsic capacity to translocate amphiphilic drugs such as Dox across the membrane via a biophysical process.     

Iodinated phospholipids and the iodination of proteins of dog thyroid gland in vitro

Slices of dog thyroid gland were incubated with liposomes consisting of (125)I-labelled phosphatidylcholine (the iodine was covalently linked to unsaturated fatty acyl chains). The (125)I label of (125)I-labelled liposomes was incorporated into thyroid protein and/or thyroglobulin at a higher rate than was the (131)I label of either Na(131)I or (131)I(2). The iodine was shown to be protein-bound by the co-migration of the labelled iodine with protein under conditions where free iodine, iodide and lipid-bound iodine were removed from protein. The uptake of iodine from the iodinated phospholipid was probably due to phospholipid exchange between the iodinated liposomes and the thyroid cell membrane, since (a) (14)C-labelled phospholipid was metabolized to (14)CO(2) and (b) many lipids in the tissue slice became (14)C-labelled. A very strong inhibition of iodide ;uptake' from Na(131)I, caused by thiosulphate, produced only a minor inhibition of the incorporation of (125)I from (125)I-labelled liposomes into thyroid protein and/or thyroglobulin. This implies that free iodide may not necessarily be formed from the iodinated phospholipids before their entrance or utilization in the cell. Synthetic polytyrosine polypeptide suspensions showed some iodination by (131)I-labelled liposomes. In tissues with low tyrosine contents, such as liver and kidney, only a trace uptake was observed. Salivary gland showed some uptake. Endoplasmic reticulum of thyroid gland showed a higher iodine uptake than that of the corresponding plasma membranes. These experiments, together with the demonstration of the diet-dependent presence of iodinated phospholipids in dog thyroid, leads us to suggest that iodination of the membrane phospholipids of thyroid cells may be directly or indirectly involved at some stage in the synthesis of thyroglobulin, or exists as a scavenger mechanism, to re-utilize and/or recover released iodine from unstable compounds inside the thyroid cell.     

Mechanisms for generating cell membrane antigens that are recognized by cytotoxic T lymphocytes

Studies with many viruses have revealed that viral specific protein synthesis is an obligatory step in generating antigens on target cells for antiviral cytotoxic T lymphocytes. This has been most clearly demonstrated with DI particles, virions that are structurally complete but lack infectious RNA. Adsorption of such particles onto target cell membranes does not render these cells susceptible to lytic attack by antiviral effector cells, unless some viral protein synthesis transpires. However, some viruses, such as Sendai virus, circumvent the requirement for viral protein synthesis via fusion of the viral envelope with the target cell membrane, a process mediated by a specialized fusion protein. Once inserted into the lipid bilayer, it is likely that viral components and self H-2 noncovalently associate so that the complex can be recognized by antiviral cytotoxic T cells. This idea is supported by the demonstration that viral proteins and H-2 containing membrane proteins, incorporated into reconstituted membrane vesicles or liposomes are recognized by cytotoxic T cells. These data further show that native rather than altered viral and H-2 molecules are the moieties recognized. Associations between antigen and H-2 have been detected by a variety of techniques and in some cases are not random but selective; that is, viral antigens perferentially associate with some H-2 alleles and not others. In summary, these findings indicate that although viral antigens are present in the mature virions, these components are not recognized by antiviral killer cells until integrated into the plasma membrane. This may be achieved either through direct fusion of the viral envelope with the target cell or following viral protein synthesis and insertion of viral antigens into the plasma membrane.     

P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir-Blodgett monolayer

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.     

P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir-Blodgett monolayer

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.