1 Département d'Ingénierie et d'Etudes des Protéines and 3 Département de Biologie Cellulaire et Moleculaire, CEASaclay, 91191 Gif sur Yvette cedex and 2 UMR 5539 CNRS, Université Montpellier II, 34095 Montpellier cedex 05, France
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Abstract |
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Keywords: diphtheria toxin/immunoglobulin/membrane anchor/protein A/transmembrane domain
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Introduction |
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Our goal is to develop an alternative strategy to gene transfer, to anchor a given protein at the surface of cells. This strategy is based on the development of a protein membrane anchor derived from the diphtheria toxin transmembrane (T) domain (Liger et al.1998; Nizard et al.1998
).
Diphtheria toxin is a 58 kDa protein organized in three domains: receptor binding (R), transmembrane (T) and catalytic (C) (Bennett and Eisenberg, 1994). After binding to its cell surface receptor, the toxin is internalized. The acidic pH in the endosome triggers the insertion of T in the membrane. This assists the translocation of C to the cytoplasm where it blocks protein translation by ADP-ribosylation of elongation factor 2 (Lemichez et al.1997
). The structure of the T domain (about 22 kDa) is organized in three layers of
-helices (Bennett and Eisenberg, 1994
): a central hydrophobic helical hairpin sandwiched and hidden from the solvent by two amphiphilic layers. Acidic pH induces a conformational change leading to exposure of the hydrophobic parts of the central layer, promoting interaction with the membrane (Zhan et al.1995
).
We have shown previously that the T domain may function as a membrane anchor to attach soluble proteins fused to its C-terminus on the surface of cells. This was done for cytokines (Liger et al.1998) and the IgG binding protein ZZ (Nizard et al.1998
). ZZ was generated by duplication of a mutated B domain from the staphylococcal protein A (Nilsson et al.1987
; Ljungberg et al.1993
; Jansson et al.1998
). The fusion protein T-ZZ, once bound to the surface of lipid vesicles or cells, was able to bind IgG, thus functioning as a membrane anchor for antibodies. Anchoring to membranes is fast and easily triggered by incubation at a mildly acidic pH (pH 5).
In the present work, we further characterized the properties of the T domain as a membrane anchor. Using the ZZ protein as a model, we studied the possibility of fusing proteins to its N-terminus or to both extremities. This was intended to establish whether it is possible to choose the orientation of the protein to be anchored, its N- or C-terminus towards the membrane. We compared the membrane binding properties of the different constructs. Finally, we investigated the fate of the molecules once attached to the surface of cells.
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Materials and methods |
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Vector pCD2-ZZ for the expression of the fusion protein T-ZZ (Nizard et al.1998) and vector pCP for the expression of the protein ZZ (Drevet et al.1997
) have been described previously. All other plasmids were derived from those. pA/T encoding the isolated T domain was obtained, first by the digestion of pCD2-ZZ with SphI and HindIII to remove the sequence encoding ZZ and then by cloning in place a DNA fragment made by hybridization of the oligonucleotides 5'-CTT AGT AAA-3' and 5'-AGC TTT TAC TAA GCA TG-3' in order to introduce two stop codons at the end of the T domain coding sequence. pA/ZZ-T encoding the fusion protein ZZ-T was constructed, first by the digestion of pCP with XmaI and BamHI to remove the 3' multiple cloning site and then by cloning in place the T coding fragment synthesized by PCR amplification from pCD2-ZZ using the primers 5'-CGG GGG TTC TGG TGG TTC TGG AGG TTC TGG TGG TTC TGG GTC TGG TTG CAT CAA CCT GGA TTG GGA-3' and 5'-GCA GCT GGA TCC TTA TCA AGC ATG CGT CTT GTG ACC C-3'. These primers were designed to introduce the restriction sites XmaI and BamHI for cloning and a sequence encoding a hydrophilic peptide spacer between ZZ and T (Table I
). Finally, pZZ-T-ZZ encoding the triple fusion protein ZZ-T-ZZ was constructed by digestion of both plasmids pA/ZZ-T and pCD2-ZZ with XbaI and ClaI located within the promoter region of the plasmids and in the T domain coding region, respectively. The fragment encoding ZZ and half of T from pA/ZZ-T was then cloned in the digested pCD2-ZZ vector carrying the remaining T coding region followed by a ZZ coding sequence.
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Expression was done in BL21(DE3) cells at 37°C. Bacteria were grown in Terrific Broth (12 g/l tryptone, 24 g/l yeast extract (Difco, Detroit, MI), 0.4% (v/v) glycerol, 17 mM KH2PO4, 72 mM K2HPO4) for proteins ZZ, ZZ-T and ZZ-T-ZZ and in M9 [5 mM (NH4)2SO4, 22 mM KH2PO4, 39 mM Na2HPO4, 39 mM NaH2PO4, 8.5 mM NaCl, 1mM MgSO4, 0.5 µM FeCl2, 0.1 mM CaCl2, 5 g/l glucose] for proteins T and T-ZZ. All media were supplemented with 0.2 mg/l ampicillin and pH was kept around 7.2 during culture. When cultures reached mid-logarithmic phase (OD600 = 0.60.7 ), protein expression was induced with 0.1 mM IPTG (Fluka, Buchs, Switzerland) carried out for 4 h. Cells were harvested by centrifugation at 4500 g for 30 min at 4°C and resuspended in 15 ml of TN buffer (50 mM TrisHCl, pH 7.8, 150 mM NaCl), excepted cells transfected with plasmid pA/T that were resuspended in 15 ml of PNI-10 buffer (20 mM sodium phosphate, pH 7.8, 0.5 M NaCl, 10 mM imidazole). After re-suspension, cells were supplemented with 0.1 ml of 0.1 M PMSF (Sigma) in ethanol and 1 ml of a 10 mg/ml solution of lysozyme (Sigma). After incubation for 1 h on ice, extracts were homogenized by repeated passage through a needle with a syringe followed by sonication for 10 min (power setting 60%, pulse 1 s, rest 1 s). The soluble cytoplasmic fraction was recovered by centrifugation at 18 000 g for 30 min at 4°C. The pellet was re-suspended in the same buffer, sonicated again and the second soluble fraction was recovered by centrifugation. Both soluble fractions were pooled and treated with 1 µg/ml of DNase I and RNase A for 1 h at 24°C and filtered through 0.45 and 0.22 µm filters. Before purification, soluble fractions were kept on ice to avoid protein degradation and aggregation.
Purification of the recombinant T domain
The T domain was purified by immobilized metal ion (Ni+) affinity chromatography (IMAC) using a HiTrap Chelating Sepharose Fast Flow gel (Pharmacia, Uppsala, Sweden). A 5 ml volume of gel was prepared according to the manufacturer's instructions. The protein sample was loaded and the column was successively washed with PNI-50 and PNI-70 buffers (20 mM sodium phosphate, pH 7.8, 0.5 M NaCl, 50 and 70 mM imidazole). Elution was done with PNI-500 buffer (20 mM sodium phosphate, pH 7.8, 0.5 M NaCl, 0.5 M imidazole). The eluted proteins were treated with 14.3 mM 2-mercaptoethanol for 1 h at 24°C to impair any intermolecular cystine formation. Then, imidazole, NaCl and 2-mercaptoethanol were removed by buffer exchange with 20 mM sodium phosphate, pH 7.8 on a Sephadex G-25 Superfine Hitrap desalting column (Pharmacia Biotech, Uppsala, Sweden). All proteins were stored in 20 mM sodium phosphate, pH 7.8 at 20°C.
Purification of the recombinant proteins ZZ, T-ZZ, ZZ-T and ZZ-T-ZZ
Purifications were conducted by affinity chromatography on an IgG Sepharose 6 Fast Flow column according to the manufacturer's protocol (Pharmacia Biotech), except for the washing buffer, the pH of which was adjusted to 4.5 instead of 5. The eluted proteins were lyophilized and stored at 20°C as such or after re-suspension in 20 mM sodium phosphate, pH 7.8.
Lipid vesicles permeation study
Large unilamellar vesicles (LUV) were prepared as described for the atomic force microscopy study below and the permeabilization study was carried out as described previously (Nizard et al.1998).
Atomic force microscopy study
LUV in PBS were prepared by reversed-phase evaporation as described (Rigaud et al.1983) using a mixture of egg phosphatidylcholine and egg phosphatidic acid (Avanti Polar Lipids, Alabaster, AL) at a molar ratio of 9:1. LUV at concentration of 2 mg/ml (2.6 mM lipids) were incubated with the fusion protein T-ZZ at a concentration of 50 µg/ml (1.3 µM) in PBS supplemented with 10 mM acetate, at a final pH of 4.8 or 7.4, for 1 h at 20°C. In controls, LUV were incubated at both pH in the absence of protein. Preparations were then deposited on mica slabs to allow the LUV to bind to the mica surface at room temperature. The slabs were then gently rinsed with water to remove unbound material and salts, which interfere with atomic force microscopy scanning, and allowed to dry. Samples were observed by scanning of 4x4 or 7.5x7.5 µm areas in non-contact mode with the tip of a Topometrix microscope.
Cell surface binding immunodetection assay
Assays on adherent L929 cells were performed in 96-well plates as described previously (Liger et al.1998; Nizard et al.1998
), except that vizualization was done as described below for the assay on A20 cells. Assays on A20 cells were done on suspensions of 6x106 cells in 15 ml Falcon tubes and all washes were done by centrifugation at 1200 r.p.m. in PBS pH 7.4. After three washes, the recombinant proteins were added to the cells at the indicated concentration in PBS supplemented with 10 mM acetate at pH 7.4 or 4.8. After incubation for 1 h at 20°C, cells were washed once and transferred to a new tube. Cells were incubated with 1.5 ml of a 1:1000 dilution of a mouse monoclonal IgG2a in PBS, pH 7.4 supplemented with 0.1% BSA for 30 min at 20°C. After three washes, cells were incubated with 1.5 ml of a 1:5000 dilution of goat
anti-mouse IgGF(ab')2peroxidase conjugate (Immunotech, Marseilles, France) in PBS, pH 7.4 supplemented with 0.1% BSA for 30 min at 20°C. After three washes, cells were re-suspended in 0.1 M TrisHCl, pH 8.5 and counted. For each sample, 2x105 cells per well were seeded in triplicate on 96-well plates (Nunc). Plates were developed with 150 µl of HPPA substrate at 26.5 mM (Sigma) diluted in 0.1 M TrisHCl, pH 8.5, 0.13% Tween-20 supplemented with 3.75/10 000 (v/v) H2O2. After 30 min at 20°C, the reaction was stopped with 50 µl of 2 M glycine, pH 10.3. Fluorescence (
ex 330 nm,
em 425 nm
) was read in a Fluorolite 1000 plate fluorimeter (Dynatech Laboratories).
When applicable, modifications of the procedure are indicated in the Figure captions. For proteinase K treatment, A20 cells were incubated with 0.05 mg/ml of proteinase K (Sigma) in PBS, pH 7.4 at 37°C for 30 min. Cells were washed three times in PBS before incubation with the fusion proteins and vizualization was performed as described above.
Confocal microscopy
Cells L929 were plated on glass cover-slips (12 mm diameter in 24-well plates) the day before the experiment. They were washed twice with PBS and once with anchoring buffer (24.5 mM sodium citrate, 25.5 mM citric acid, pH 4.7, 280 mM sucrose) before incubation for 30 min at room temperature with T-ZZ or ZZ-T at 106 M in 0.4 ml of anchoring buffer. Control cells were treated with anchoring buffer only. For some experiments, cells were washed with PBS and cultivated for 24 h before transferrin incubation and processing for immunofluorescence. After two washes with PBS, cells were incubated with transferrin-FITC (25 µg/ml in DMEM containing 0.1 mg/ml BSA) for 45 min at 37°C. They were then cooled to 4°C, washed once with DMEMBSA and twice with PBS. Cells were subsequently fixed with 3.7% paraformaldehyde (freshly prepared in PBS) for 30 min at room temperature. The fixative was quenched for 10 min with 50 mM ammonium chloride in PBS and the cover-slips were transferred to a humidified chamber and incubated for 45 min in permeabilization buffer (PBS supplemented with 0.02% saponin and 1 mg/ml BSA) containing rabbit anti-goat IgG (Nordic). After rinsing, the presence of the first antibody was revealed by incubation under the same conditions with a TRITC-labeled goat anti-rabbit IgG (Sigma). The cover-slips were then rinsed extensively with permeabilization buffer, then with PBS and briefly with water before mounting and examination under a Leica confocal microscope. Median optical sections were recorded with a x63 lens.
K562 cells growing in suspension were labeled with T-ZZ or ZZ-T and transferrin-FITC at a density of 5x106 cells/ml before processing for immunofluorescence (Subtil et al.1997) using the antibodies indicated above.
Cell permeabilization study
Vero cells were seeded on 96-well plates (Nunc) at a density of 105 cells/well in Dulbecco-MEM supplemented with 10% fetal calf serum (FCS) and 2 mM glutamine and were grown overnight to confluence. Cells were washed three times with medium without FCS. Then 200 µl of BCECF-AM solution (50 µg of lyophilized BCECF-AM (Molecular Probes) diluted with 72 µl of DMSO and 7.2 ml of medium without FCS) were added. Cells were then incubated for 1 h and washed three times with medium without FCS
. A 50 µl volume of PBS, pH 7.4 was added to the cells and a first fluorescence measure (ex 465 nm,
em 530 nm
) was done to define 100% fluorescence, using a Fluorolite 1000 plate fluorimeter. Medium was removed and recombinant proteins were added at the indicated concentrations in 50 µl of PBS adjusted to pH 4.8 with H2SO4. Fluorescence was monitored for 18 min. Then 0.5 µl of 1 M acetate, pH 3.6, which diffuses across cell membranes, was added (the final pH in the well remained at 4.8) to acidify the cytoplasm of cells. Fluorescence was further monitored for 5 min.
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Results |
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We have produced three fusion proteins, in which the ZZ protein was fused to either the N- or C-terminus or to both extremities, of the T domain. These constructs are referred to as T-ZZ, ZZ-T and ZZ-T-ZZ. We have also produced the isolated T domain and ZZ protein for controls. Table I summarizes the primary structure of all five molecules. The 10 last amino acids of the T domain (from Arg377 to Thr386) are stretched out of its globular structure (Bennett and Eisenberg, 1994
). In the T-ZZ protein, they constitute a spacer between the two fusion partners, together with two additional residues. In ZZ-T and ZZ-T-ZZ, a flexible hydrophilic peptide spacer rich in Gly and Ser residues was inserted between ZZ and T.
After expression and purification of the proteins, the final recovery yields ranged from 10 mg for T to 60 mg for ZZ-T-ZZ per liter of bacterial culture. SDSPAGE and silver staining of the recombinant proteins showed that they had been purified nearly to homogeneity (Figure 1). They have an electrophoretic mobility consistent with the expected molecular masses deduced from their amino acid sequences: 16.74 kDa (149 residues) for ZZ, 22.01 kDa (201 residues) for T, 36.38 kDa (328 residues) for T-ZZ, 36.29 kDa (335 residues) for ZZ-T and 50.66 kDa (462 residues) for ZZ-T-ZZ. All proteins containing the ZZ part were capable of binding IgG. This was demonstrated by IgG affinity chromatography, the procedure used for their purification.
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Interaction with membranes of the isolated T domain and related fusion proteins, as well as native diphtheria toxin, can be assessed in an assay measuring the release of a fluorescent dye entrapped into lipid vesicles (Zhan et al.1995; Liger et al.1998
; Nizard et al.1998
; Sharpe et al.1999
). All proteins containing the T domain were able to permeabilize LUV at acidic pH (Figure 2
)
, whereas no permeabilization occurred at pH 7.4 (not shown). The isolated ZZ protein had no effect at either pH. Comparison of the effect of increasing concentrations of each protein shows that they all have comparable permeabilization efficiency on a molar basis and that this effect is dose dependent (Figure 2B
). These results indicate that the capacity of the T domain to interact with membranes is not altered when another protein is fused to its N- or C-terminus or to both extremities.
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We compared the binding capacities of the fusion proteins to the surface of L929 fibroblasts (Figure 4A and B)
and A20 cells (Figure 4C and D
). L929 cells attach to the plastic of the tissue culture plates, on to which they proliferate to confluence. A20 cells grow in suspension. The cells were incubated with varying concentrations of the fusion proteins at pH 4.8 for 1 h at 20°C. Binding to the cells was measured by immunoenzymatic detection using a mouse monoclonal IgG2a, capable of binding to ZZ by its Fc part and a goat anti-mouse IgGF(ab')2peroxidase conjugate. The results show that the fusion proteins T-ZZ and ZZ-T have the same capacity to bind to the surface of cells, in a dose-dependent manner. The same result was found for ZZ-T-ZZ with L929 cells. However, this triple fusion protein contains two ZZ moieties and should theoretically bind twice as much IgG as the two other fusion proteins. We found previously that all three proteins have the same capacity to interact with LUV. Thus, two possible explanations could account for this: either one of the ZZ moieties is masked by the other one within the bound ZZ-T-ZZ molecule, or once an IgG is bound to one ZZ it masks recognition of the second ZZ moiety by another IgG.
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Overall, these results show that the T domain may function as a membrane anchor for the ZZ protein fused at its N- or C-terminus. The capacity of these anchors to attach IgG to cells does not depend on the extremity of the T domain to which ZZ is fused. However, although T can be used to anchor two ZZ domains fused to both of its extremities, this does not lead to an increase in the amount of IgG anchored to cells. Optimal binding occurs for concentrations within the µM range using incubations of 1 h. This incubation time was chosen according to the kinetic study described in the next section.
Kinetics of binding to cells
We studied the influence of incubation time on the amount of fusion proteins bound to the surface of cells. L929 fibroblasts were incubated with the fusion proteins at pH 4.8 for various times before detection. The results in Figure 5A show that maximum binding is reached after 1 h. Half-maximum binding is reached between 10 and 20 min for T-ZZ and ZZ-T and after 30 min for ZZ-T-ZZ.
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Our aim was to investigate the fate of the fusion proteins bound to the surface of live cells. L929 fibroblasts were treated with the fusion proteins at acidic pH for 1 h at 20°C, to allow binding of the proteins to the surface of the cells. The cells were then washed and placed back into culture medium containing FCS, at 37°C, in the CO2 incubator. The presence of the fusion proteins remaining at the surface of the cells was revealed after various times. Figure 5B shows that about 75% of the proteins were still displayed at the cell surface after 24 h.
Confocal microscopy was then used to study the subcellular localization of the fusion proteins. L929 fibroblasts were incubated with T-ZZ or ZZ-T at acidic pH for 30 min, washed and then incubated with FITC-labeled transferrin for 45 min. For confocal observations, the cells were fixed, permeabilized and the fusion proteins were detected using a rabbit anti-goat IgG and a TRITC-labeled goat anti-rabbit IgG. Transferrin is known to be rapidly internalized through the clathrin-coated pathway and to accumulate in early endosomes (Subtil et al.1997). TransferrinFITC was therefore used as an endosome marker. Figure 6AC
shows that transferrin (green) is internalized and localized to the perinuclear area, as expected (Subtil et al.1997
). Identical images were obtained when the transferrin receptor was revealed by immunofluorescence on permeabilized cells (not shown), indicating that the permeabilization procedure was adequate. Nevertheless, the T-ZZ and ZZ-T molecules (red) were observed at the cell surface and were not internalized.
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Similar images were obtained when a T domaincytokine fusion protein was anchored on to RMA lymphoma cells, B16 melanoma cells, and L929 cells (unpublished results). Thus, it seems that persistence of T domain fusion proteins at the surface of live cells is a general phenomenon. However, the study of the binding of the fusion proteins T-ZZ and ZZ-T on the K562 human myeloid leukemia cell line gave different results. In this case, the fusion proteins were internalized and gathered into the same compartments as transferrinFITC, as shown by the yellow color resulting from green and red dye co-localization (Figure 6G and H). This compartment is presumably the early endosome. A fraction of the proteins remained at the surface of the cells after 45 min (Figure 6G and H
). Thus, internalization of the proteins seems slower than receptor-mediated internalization of transferrin. No protein was detectable either at the surface or into cell compartments after 24 h (not shown).
Overall, our results show that the fate of T fusion proteins bound to the surface of cells may depend on cell type. On cells that do not internalize the T fusion proteins, they remain displayed at the cell surface for more than 24 h, without much loss. On cells that do internalize the fusion proteins, they disappear from the surface more slowly than transferrin bound to its receptor and seem to gather in the early endosomes in the same manner as transferrin. After 24 h, no protein remains detectable on these cells.
Cell surface proteins are not required for binding of the fusion proteins to the cell membrane
So far, we have found that the fusion proteins described in this work, as well as T-cytokine fusion proteins, could bind to any cell type. Up to nine different cell types have been tried, giving similar results. These include fibroblasts, leukemia cell lines from various lineages, breast cancer and melanoma cell lines. This makes sense if one considers that the T domain has affinity for phospholipid bilayers. However, we investigated the possible implication of cell surface proteins in the binding of T-fusion proteins to cells. Cells were treated with proteinase K to remove extracellular domains of membrane proteins. The data in Figure 7A show that T-ZZ at acidic pH has the same capacity to bind to cells whether treated or not with proteinase K. This strongly suggests that the extracellular domains of cell surface proteins do not influence binding of T to the lipid bilayer of the membrane.
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We have shown previously that the interaction of the T domain and its related fusion proteins, with LUV at acidic pH modifies their permeability dramatically (Figure 2). It was therefore important to investigate whether the T domain fusion proteins alter the permeability of cell membranes. Indeed, it has been shown that interaction of the complete diphtheria toxin with the membrane of Vero cells at acidic pH could lead to the formation of ion channels under certain conditions (Papini et al.1988
; Sandvig and Olsnes, 1988
; Stenmark et al.1988
). These channels were found to be selective for small cations such as H+, Na+ and K+. To examine this question, Vero cells were loaded with BCECF, a fluorescent intracellular pH probe, using the acetoxymethyl ester loading method. The cells were incubated with the fusion proteins at pH 4.8 and their fluorescence was monitored. In case of increased membrane permeability, H+ ions would enter the cells and acidify their cytoplasm, leading to a decrease in fluorescence. In the absence of protein, a loss of fluorescence intensity of 25% was observed, indicating a moderate acidification of the cytoplasm of the cells (Figure 7B
). No further acidification of the cytoplasm was observed in the presence of any of the T fusion proteins or the isolated T domain over 12 min. As a positive control for cytoplasm acidification, an acetate buffer at pH 4.8 was used. Acetate, diffusing through the cell membrane, resulted in a 50% decrease in fluorescence intensity within 3 min. Overall, these results strongly suggest that binding of the T domain fusion proteins, as well as the isolated T domain, at acidic pH, does not increase cell membrane permeability to H+. In addition, no alteration of cell shape or cell membrane bursting was ever observed by microscopy following this treatment.
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Discussion |
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The N-terminal side of T plays an important role in the translocation process of the C domain within the native toxin (Madshus, 1994; Madshus et al.1994
). However, no membrane insertion or translocation of ZZ seems to occur when anchoring the fusion protein ZZ-T to cells. Indeed, its ZZ part is recognized by IgG in a similar fashion as for T-ZZ. It has been shown previously that binding of the toxin to its receptor was a prerequisite for C domain entry into cells from the cell surface at acidic pH (Stenmark et al.1988
; Madshus et al.1991
). In our case, no protein receptor is involved in the interaction with the membrane. In addition, C must change its conformation in order to interact with T, insert into the membrane and translocate (Falnes and Olsnes, 1995
; Ren et al.1999
). ZZ, being very stable and soluble (Samuelsson et al.1994
), is not suited to membrane interaction or translocation.
A striking result is that whereas lipid vesicles are destabilized by the interaction of the membrane anchors (Figure 3), the cell membrane seems not to be affected. Cells remain fully viable (Nizard et al.1998, and unpublished results
), their membranes are not permeabilized (Figure 7
), no alteration of cell morphology was observed upon confocal microscopic examination (Figure 6
) and in atomic force microscopy studies of cell surface (not shown). This is perhaps not surprising. The cell membrane is far more complex than the membrane of LUV. It has a very different lipid composition including cholesterol, leaflet asymmetry and transmembrane domains of proteins and is connected to a cytoskeleton. The fusion of lipid vesicles induced by diphtheria toxin at acidic pH has already been described using fluorescence resonance energy transfer (Papini et al.1987
). Here, we illustrated this phenomenon by showing the extent of LUV structure alteration (Figure 3
). This result may be interesting for those studying the effect of hemolytic toxins on lipid vesicles, as the nature of the lesions in the membranes of cells or vesicles may be very different.
Finally, we found that the fate of the fusion proteins anchored to the surface of cells may differ, depending on the cell type. On fibroblasts (this study), lymphoma cells and melanoma cells (unpublished results), the proteins remain associated with the surface of the cells for at least 24 h without much loss. In sharp contrast, K562 cells internalize the proteins, almost as efficiently as transferrin. So far, we have studied binding of T-derived fusion proteins to the surface of nine different cell lines and have not found important differences in binding levels. Although it is necessary to check, for any new cell type, the behavior of the fusion proteins, it is possible that most cells will not internalize them. This difference in behavior may lead to different applications for the use of T domain membrane anchors. In the case where fusion proteins remain at the plasma membrane, it is possible to display a protein at the surface of cells for maybe several days. This protein could act as a signal for or promote interaction with other cells. It could also be used as a marker to follow a labeled cell in a population. The pathway through which some cell types internalize the fusion proteins could be used to deliver such proteins to the endosomes. However, this pathway will need further characterization.
Other membrane anchors have been described to attach proteins to cells. Chelator lipids can be incorporated into the cell membrane to attach histidine-tagged proteins using nickel ions (van Broekhoven et al.2000). A glycosylphosphatidylinositol anchor (McHugh et al.1999
) and an acylated tag anchor (de Kruif et al.2000
) have also been described. Each system may have its own advantages depending on the application, the ease of tagged protein expression and purification, the conditions of anchoring needed (dead or live cells, need for a triggered anchoring such as the pH for T), the persistence on the cell surface, etc.
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Notes |
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Acknowledgments |
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Received November 20, 2000; revised March 3, 2001; accepted March 20, 2001.