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1 Research Institute, Program in Molecular Structure and Function, The Hospital for Sick Children, Ontario M5G 1X8, Canada
2 Departments of Laboratory Medicine and Pathobiology, University of Toronto, Canada
3 Department of Biochemistry, University of Toronto, Canada
Correspondence
Clifford A. Lingwood
cling{at}sickkids.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Supplementary material showing quantitation of VT1 A1 translocation and extrapolation to the CD50 is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Ingestion of EHEC can result in haemorrhagic colitis that can progress to the haemolytic uraemic syndrome (HUS) (Ochoa & Cleary, 2003). A direct association has been established between the development of HUS and verotoxins (also termed Shiga toxins) produced by EHEC (Karmali, 2004; Karmali et al., 1983). Verotoxins are AB5 subunit toxins, possessing catalytic A subunits and receptor-binding B subunits. Verotoxin 1 (VT1) is one of the two major variants involved in human disease, and contains one 32 kDa A subunit (VTA) and a 7.7 kDa B subunit pentamer (VTB). VTA and VTB are secreted into the bacterial periplasm, where they assemble non-covalently into holotoxin.
The VT1 receptor is a glycosphingolipid, globotriaosylceramide (Gb3) (Lingwood, 1993). The VT1–Gb3 complex is internalized by receptor-mediated endocytosis (Khine & Lingwood, 1994; Sandvig et al., 1989), both clathrin and caveolae dependent (Nichols et al., 2001). Following kinase-dependent endocytosis (Lauvrak et al., 2006), VT1 exploits a retrograde transport route to the Golgi and endoplasmic reticulum (ER) (Sandvig et al., 1992). This Golgi/ER retrograde transport pathway has been characterized using VTB (Johannes et al., 1997), and sorting along this pathway is also clathrin dependent (Lauvrak et al., 2004; Saint-Pol et al., 2004).
The ribosome-inactivating function of VTA has been well characterized (Obrig et al., 1987; Saxena et al., 1989). During transport, VTA must undergo disulfide bond reduction and proteolysis at a C-terminal furin-sensitive site (Garred et al., 1995) to produce the active A1 fragment (VTA1) (Lea et al., 1999). The current model for verotoxin internalization suggests that following intracellular processing, VTA1 is translocated to the cytosol from the ER (Sandvig & van Deurs, 2002). VT1 can be coprecipitated with ER chaperones (Falguieres & Johannes, 2006; Yu & Haslam, 2005), and two independent studies have shown in situ that translated VTA can be translocated across the yeast ER membrane (LaPointe et al., 2005) and purified microsomal membranes (Yu & Haslam, 2005). However, this translocation has yet to be demonstrated in Gb3-expressing cells when VTA has undergone internalization, retrograde transport, cleavage, reduction and separation from VTB.
While VTA may have a role in the upregulation of clathrin-dependent VT1 internalization (Torgersen et al., 2005), few studies have focused on VTA. To avoid the cytotoxic effects associated with VTA, most VT1 transport studies have utilized VTB (Arab & Lingwood, 1998; Johannes et al., 1997). Although some studies have monitored VT1 as a whole (Sandvig & van Deurs, 1996), no studies have monitored both VTA and VTB. Consequently the VTA–VTB association during retrograde transport is unknown.
The present work focuses on VT1 holotoxin, specifically VTA transport in relation to VTB using confocal fluorescence microscopy, translocation of VTA1 to the cytosol, and the relationship of VTA1 translocation to inhibition of protein synthesis and cytopathology.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Anti-Rab6 and -GRP78 antibodies were from Santa Cruz Biotechnology. Anti-protein disulfide isomerase (PDI) antibody was from QED Bioscience; anti-Hsc70 antibody was from Stressgen Biotechnologies. FITC, tetramethylrhodamine (TAMRA) and Alexa-conjugated antibodies were from Molecular Probes. Cy5-conjugated antibodies were from Jackson ImmunoResearch Laboratories. Lactacystin, brefeldin A (BFA) and streptolysin O (SLO) were from Sigma. In all experiments with BFA or lactacystin, drug concentrations were maintained throughout the experiment.
Cell culture.
Vero cells were maintained in alpha-Modified Eagle's Medium (MEM) with 5 % fetal calf serum.
Preparation of VTA/VTB dual-labelled holotoxins.
VT1 was purified as described by Nutikka et al. (2003). VT1 was coupled to TAMRA or FITC as per the manufacturer's instructions. One milligram of TAMRA- or FITC-labelled VT1 in PBS was concentrated to 50–100 µl in a Centricon-30 microconcentrator (Millipore). The concentrate was diluted to 1 mg ml–1 with separation buffer (6 M urea, 4 M guanidine, 0.1 M propionic acid, 150 mM NaCl, pH 4) and placed at 4 °C for 2 h for subunit denaturing and separation. VTA and VTB were separated by HPLC on a TSK-G2000SW gel filtration column (7.5 x60 mm; Supelco) equilibrated with separation buffer (Head et al., 1991; Ito et al., 1988). Subunit purity was verified by SDS-PAGE.
To refold active VT1, FITC–VTA and TAMRA–VTB were mixed and dialysed overnight into 200 mM potassium phosphate, 0.5 mM CHAPS, pH 7.2, at 4 °C, using 3.5 kDa molecular weight cut-off (MWCO) dialysis tubing (Spectra/Por). Refolded VT1 was isolated from free subunits by HPLC (200 mM potassium phosphate, 0.5 mM CHAPS, pH 7.2, flow rate 0.75 ml min–1). Following SDS-PAGE, the subunit fluorescence of the refolded dual-labelled VT1 was verified under UV light.
Generation of polyclonal anti-VTA.
VTA was isolated as described above. The level of residual active VT1 was <0.01 % by Vero cell cytotoxicity. A New Zealand white rabbit was immunized subcutaneously with 200 µg VTA emulsified with Freund's complete adjuvant and boosted 3 weeks later with 200 µg VTA in Freund's incomplete adjuvant. Two weeks post-boost, serum was prepared and tested for the VTA-specific antibodies by Western blotting.
Verotoxin cell cytotoxicity assay.
The bioactivity of the refolded VT1 was compared with that of unmodified VT1. Vero cells seeded in 96-well plates (20 000 cells per well) were grown overnight. Tenfold serial VT1 dilutions were added at 37 °C. Cell viability was assessed by crystal violet staining after 72 h and the VT1 dose killing 50 % of the cells (CD50) was measured from the curve.
Immunocytochemistry and fluorescence microscopy.
For immunostaining and direct-labelled VT1 staining, cells were grown on 12 mm glass coverslips to 
80 % confluence. Cells were incubated with unlabelled VT1 or dual-labelled VT1 at 1 µg ml–1, in media, on ice for 30 min. After washing, cells were incubated at 37 °C. Cells were fixed with 4 % paraformaldehyde (PFA) in PBS. Coverslips were mounted in Dako mounting medium. For immunostaining, following 4 % PFA fixation, cells were permeabilized with saponin solution (0.05 % saponin, 0.1 % BSA in PBS). All antibody and wash steps were performed in saponin solution. Anti-Rab6 or -GRP78 antibody (1 : 100 dilution) was added for 1 h; cells were then washed three times for 5 min. Anti-Cy5 or -Alexa secondary antibody (1 : 250 dilution) was added for 1 h. Cells were washed three times for 5 min, fixed in 4 % PFA, and mounted in Dako mounting medium. Fluorescently stained cells were viewed with a Zeiss LSM510 confocal system. Images were processed with Adobe Photoshop and Zeiss Image Examiner.
Measurement of protein synthesis.
Cells were seeded in 24-well plates (50 000 cells per well) for 48 h. For experiments with BFA, cells were preincubated with 2.5 µg BFA ml–1 for 30 min prior to toxin binding. Cells were incubated with 1 µg VT1 ml–1 for 30 min on ice, then washed with PBS. VT1 was internalized for 1 or 6 h at 37 °C. Incorporation of [3H]leucine (1 µCi ml–1; 37 kBq ml–1) was measured for 30 min at 37 °C. Cells were washed with PBS, and proteins were precipitated with cold 10 % TCA and solubilized in 0.1 M KOH. Protein synthesis was calculated as the percentage of incorporated [3H]leucine compared with that of control cells.
For experiments with lactacystin, cells were preincubated with 5 µM lactacystin for 90 min prior to toxin addition. VT1 was internalized continuously for 4 h at 37 °C prior to [3H]leucine incorporation.
For protein synthesis measurements at 16 °C, cells were pretreated with or without lactacystin or BFA and then treated with 1 µg VT1 ml–1 or 0.5 µg VTB ml–1 for 3 h at 16 °C. Protein synthesis was measured for 1 h in leucine-free Dulbecco's MEM containing 25 mM HEPES and 10 µCi ml–1 (370 Bq ml–1) [3H]leucine.
Cell permeabilization with SLO.
VT1 was radiolabelled with Na125I in Iodogen-coated tubes (Pierce). Vero cells were grown in six-well plates for 48 h (80 % confluence). [125I]VT1 (1 µg ml–1) was internalized for 4 h at 37 °C. Cells were placed on ice and washed extensively with PBS. SLO (250 U ml–1) was added for 10 min on ice, in SLO buffer (115 mM potassium acetate, 2.5 mM MgCl2, 25 mM HEPES, pH 7.5). Cells were washed three times with SLO buffer to remove unbound SLO. SLO buffer (1 ml) with protease inhibitors (Complete protease inhibitor tablets, Roche) was added to cells. Cells were transferred to 37 °C for 15 min for plasma membrane pore formation. Permeabilized cells were put on ice for 30 min to allow cytosol to leak out. Cell debris was removed by high-speed centrifugation, and the supernatant was used for immunoprecipitation.
To verify that organelle membranes remained intact during SLO permeabilization, Western blots were performed on the cytosolic fractions. Cytosol fractions were concentrated, separated by 10 % SDS-PAGE, and transferred to nitrocellulose. Blots were blocked in 5 % milk/Tris-buffered saline, then probed with antibody for 1 h. Anti-PDI was used to probe for organelle leakage, and anti-Hsc70 was used to confirm plasma membrane permeabilization. Horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma) was added for 1 h, and bands were detected by ECL (Pierce).
Immunoprecipitation.
Prior to immunoprecipitation (IP) of cytosolic VTA1, the cytosol supernatants were pre-cleared with an initial IP with anti-PH1 antibody bound to protein A/G agarose (Amersham Pharmacia) to remove residual holotoxin. Residual VT1 was present in the supernatant fractions from both SLO-permeabilized and non-permeabilized cells. As the plasma membrane is intact with buffer alone, this VT1 must reflect residual cell-surface-bound toxin.
IP of cytosolic VTA1 from the supernatant was performed using anti-VTA bound to protein A/G agarose. IP was performed on a rotating shaker at 4 °C overnight followed by washing with IP buffer (0.5 % Triton X-100 in PBS). Immunoprecipitates were separated by non-reducing 15 % Tricine SDS-PAGE, and radiolabelled subunits were detected by a PhosphorImager (Molecular Dynamics). Band intensities were measured with ImageQuant software (Molecular Dynamics).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, cytotoxicity curves for the dual-labelled and unlabelled toxins are completely overlapping, which shows that the dual-labelled VT1 retained full bioactivity. Thus, dual-labelled VT1 was an appropriate tool to monitor the intracellular transport of VTA and VTB.
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). After 1 h at 37 °C, both VTA and VTB colocalized with the Golgi marker Rab6, although Rab6 staining was somewhat more restricted, possibly due to additional staining of the Golgi/ER transitional elements by VT1. At 1 h, internalized unlabelled VT1 detected by indirect immunofluorescence was also colocalized with Rab6 (Fig. 2b), and in a similar manner to dual-labelled VT1, unlabelled VT1 was less restricted within the Golgi than Rab6. Thus, Golgi transport of both dual-labelled VT1 (Fig. 2a) and unlabelled VT1 (Fig. 2b) corresponded with transport of mono-labelled VT1 and VTB (Heath-Engel & Lingwood, 2003; Johannes et al., 1997; Sandvig et al., 1992; Schapiro et al., 1998; White et al., 1999).
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). Within the limits of confocal microscopy, no VT1 subunit separation was apparent; cytosolic VTA could not be detected.
Our findings show that VTA is cotransported with VTB to the Golgi and ER. Visualization by confocal microscopy requires 1 µg VT1 ml–1, a concentration far higher than the Vero cell CD50 (5 pg ml–1; see Fig. 1). Thus, this assay can only monitor the bulk of internalized toxin. A small fraction of VTA within the cytosol, separate from VTB, might go undetected.
Cytosolic translocation of [125I]VTA1
To detect cytosolic VTA1, a radiolabelled VT1, [125I]VT1, was used as a more sensitive probe. SLO was used to selectively permeabilize cell plasma membranes to allow cytosol collection (Rapak et al., 1997). First, we ensured that SLO permeabilized the plasma membrane but not the ER membrane. Fig. 3 shows that Hsc70, a cytosolic protein, is detected in the cytosol fraction of SLO-permeabilized cells, but that PDI, a lumenal ER-restricted protein, is not detected. Hsc70 was not detected in the supernatant of non-permeabilized control cells. Thus, only the plasma membrane is permeabilized by SLO treatment, allowing collection of the selectively extruded cell cytosol. This technique was then employed on [125I]VT1-treated cells for the detection of any translocated cytosolic [125I]VTA1.
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). From a time-course (Fig. 4a), it was apparent that cytosolic transit was maximal at 6 h. Only the cleaved, reduced and VTB-separated [125I]VTA1 could be immunoprecipitated from the cytosol of SLO-permeabilized cells. Cytosolic [125I]VTA1 was not detected without SLO treatment. Trace cytosolic [125I]VTA1 could be detected after 1 h, increasing after 2 h to reach a plateau between 4 and 6 h of toxin exposure (Fig. 4a). In order to detect translocation, cell treatment with 1 µg [125I]VT1 ml–1 was necessary. Thus, these translocation results directly relate to the VT1 transport seen in Fig. 2(a). At 1 h, VT1 was in the Golgi (Fig. 2a) and little translocation was detected (Fig. 4a). A small fraction of VT1 that rapidly reaches the ER within 1 h (Johannes et al., 1997; Sandvig et al., 1992) would explain the low translocation at this time. VT1 accesses the ER within 3–6 h (Khine et al., 2004), and [125I]VTA1 translocation reached a maximum at this time. Since the detected VT1 subunits remained associated throughout retrograde transport from the Golgi to the ER (Fig. 2a), the time-course for translocation indicated that the ER is the likely compartment for cytosolic access. We calculated that at this concentration, only 4 % of VTA1 internalized into Vero cells was translocated to the cytosol within 4 h of continuous toxin exposure, a fraction unlikely to be detected by confocal microscopy. Extrapolation from these data, assuming a linear dose–response, gave a value of 0.75 VTA1 molecules translocated per cell at the CD50 (see supplementary material). Thus, this calculation predicts that approximately one molecule, the theoretical minimum, translocated into the cytosol would be required to kill a cell.
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). The lysate profile did not change with time and a representative sample at 4 h is shown. The same results were obtained in BFA-treated cells (Fig. 4b), indicating that VTA proteolytic cleavage occurs before VT1 reaches the Golgi, and that disulfide bond reduction occurs after VT1 reaches the Golgi. The translocated cytosolic VTA1 represented only a small percentage of the total cell-associated VT1, undetectable in the cell lysate in Fig. 4(b). Since no full-length VTA was detected in the cytosol (Fig. 4a), and most cell-associated VTA remained non-reduced (Fig. 4b), reduction of VTA, separation from VTB and cytosolic translocation must occur in rapid succession.
Role for ER-associated degradation?
Other AB subunit toxins, such as ricin and cholera toxin, translocate to the cytosol in association with the ER-associated degradation (ERAD) pathway (Deeks et al., 2002; Rodighiero et al., 2002; Wesche et al., 1999), in which misfolded proteins are translocated for proteosomal degradation (Werner et al., 1996). To assess the possible involvement of the ERAD pathway, the effect of lactacystin proteasomal inhibition on VTA1 translocation was tested. Lactacystin increased the immunoprecipitated cytosolic [125I]VTA1 (Fig. 4c, lane 4) by 30 % (monitored by counting the eluted band), suggesting that the translocated [125I]VTA1 is degraded by the proteasome/ERAD. Increased cytosolic VTA1 was accompanied by a 50 % increase in protein synthesis inhibition (Fig. 5a) and a 50 % increase in the cell cytotoxicity of VT1 (Fig. 5b). This was not due to increased cell binding by the toxin (not shown). Lactacystin gave a similar increase in the cell cytotoxicity of VT1 and ricin (Fig. 5b), implying a similar (ERAD?) translocation mechanism.
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), and collapses Golgi and ER (Lippincott-Schwartz et al., 1989). BFA protects VT1-treated cells from apoptosis (Fujii et al., 2003) and protein synthesis inhibition (Donta et al., 1995; Khine et al., 2004). BFA presumably protects cells through its collapse of the Golgi, thereby preventing further retrograde transport to the ER, from which translocation is presumed to occur. During BFA treatment, VT1 accumulates in the fused TGN/endosome (Khine et al., 2004), unable to proceed further. At low temperature (<20 °C), retrograde transport is also prevented and internalized cargo accumulates in endosomes, and transport to the lysosomes, TGN and Golgi is inhibited (Baravalle et al., 2005; Mallard et al., 1998; Punnonen et al., 1998).
Both conditions (16 °C, BFA) prevented detectable VTA1 cytosolic transfer (Fig. 4c, lanes 8 and 6). By confocal microscopy, VTA and VTB subunits remained together in fused TGN/endosomes, marked by transferrin, in BFA-treated cells at 37 °C (Fig. 6a). At 16 °C, VT1 holotoxin was internalized only to endosomes, largely colocalized with transferrin and completely distinct from the Golgi, as defined by Rab6 (Fig. 6b). Prolonged incubation did not result in further retrograde transport and no subunit separation was seen (Fig. 6c).
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). Protein synthesis was inhibited early after VT1 treatment. At 1 h, when the greater part of VTA and VTB was colocalized in the Golgi (Fig. 2a), protein synthesis was inhibited by 90 % (Fig. 7a), and a very low level of cytosolic [125I]VTA1 was detected (Fig. 4a). At 6 h, when VTA and VTB were localized in the ER (Fig. 2a), protein synthesis was inhibited by 95 % (Fig. 7a), and cytosolic [125I]VTA1 increased to maximal levels (Fig. 4a).
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). Although protein synthesis was inhibited by 50 % after 4 h at 16 °C (Fig. 7b) and after 6 h with BFA (Fig. 7a), no cytosolic VTA1 was detected (Fig. 4c, lanes 6 and 8). This protein synthesis inhibition under endosomal block could be explained by undetected VTA that separates and translocates from the endosome (as suggested for ricin; Beaumelle et al., 1993). Alternatively, the protein synthesis inhibition may be translocation independent, or undetected VT1 may reach the ER and translocate in the presence of BFA or at 16 °C (Llorente et al., 2003).
As demonstrated with BFA (Donta et al., 1995; Khine et al., 2004), higher VT1 concentrations (Fig. 7b) and longer incubation times (Fig. 7a) were required to inhibit protein synthesis at 16 °C compared with the Golgi-dependent 37 °C pathway. To further define protein synthesis inhibition at 16 °C, the effects of BFA, lactacystin and VTB on protein synthesis inhibition were examined. By confocal microscopy, the combination of 16 °C and BFA inhibited internalization of VT1 and transferrin (not shown). As a result, no protein synthesis inhibition was seen (Fig. 7c). Unlike at 37 °C, VT1-induced protein synthesis inhibition at 16 °C was not enhanced by lactacystin treatment (Fig. 7c). This suggests that the protein synthesis inhibition observed at 16 °C does not require cytosolic translocation. Inhibition of protein synthesis at 16 °C was not triggered by toxin–receptor binding/internalization, since VTB had no effect (Fig. 7c). Therefore, this effect is dependent on VTA, possibly modulated by the influence of VTA on VTB interaction at the cell surface and/or during internalization (Torgersen et al., 2005).
Inhibition of protein synthesis increases with VT1 exposure time (Fig. 7d). Only measurement of protein synthesis inhibition after >3 h toxin exposure accurately reflected subsequent cytotoxicity (Fig. 7d). Therefore, inhibition of protein synthesis at 3 h and earlier could be considered ‘overkill’, unnecessary for cytotoxicity and of questionable significance to verotoxin cytopathology.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). For the first time in intact cells, we directly demonstrate that 4 % of internalized activated VTA1 separates from VTB and is translocated to the cytosol. Previous VTA translocation studies have demonstrated that in situ translated VTA and VTA1 translocate into the cytosol, and can be immunoprecipitated with translocon components and ER chaperones (Falguieres & Johannes, 2006; LaPointe et al., 2005; Yu & Haslam, 2005), providing support for the ER translocation. However, we were unable to detect cytosolic VTA or to coimmunoprecipitate the translocon component Sec61
with VTA1 (not shown).
Linear extrapolation of our quantitative [125I]VTA1 translocation data gave a value of about one VTA1 unit per cell cytosol (the theoretical minimum) at the lowest VT1 cytotoxic dose, indicating that the translocation we observed is likely of pathophysiological significance. Cytosolic translocation is a relatively infrequent (4 %), rate-limiting step in VT1 cell cytotoxicity. Since VTA1 is not detectable in the cell lysate, the reduction of the proteolytically cleaved VTA could be rate limiting for translocation, i.e. subunit separation and translocation perhaps occur as a continuous coordinated process. In a yeast model, the C-terminal 11 amino acids of the VTA1 fragment are necessary for ER translocation (LaPointe et al., 2005) and are proposed to represent a ‘misfolded domain’ recognized by ER chaperones.
Because of the low lysine content of the A subunits of AB subunit toxins, they may escape ubiquitination and proteasome degradation (Hazes & Read, 1997). However, about 30 % of VTA1 remains susceptible to proteasomal degradation, as demonstrated by the increased cytotoxicity and cytosolic translocation of VT1 (our results) and the threefold increase in ricin potency and cytosolic translocation (Deeks et al., 2002; Wesche et al., 1999), in the presence of lactacystin. As translocated VTA1 (our results) and ricin A chain (Deeks et al., 2002) do not display increased molecular mass, indicative of ubiquitin ligation, they are likely targeted to the proteasome by ubiquitin-independent mechanisms (Kalejta & Shenk, 2003; Lee et al., 2004). For cholera toxin, evasion of proteasomal degradation is due to both low lysine content and rapid refolding in the cytosol (Rodighiero et al., 2002).
The dual-labelled VT1 holotoxin retained full cytotoxic activity and was transported intracellularly in a manner indistinguishable from that of unlabelled VT1. By far the greater portion of dual-labelled fluorescent VT1 does not separate into its component VTA and VTB subunits during retrograde transport to the ER. Since VTA and VTB remain colocalized in the ER, and [125I]VTA1 cytosolic detection coincided with ER arrival, it is likely that subunit separation and translocation occur from this compartment, in agreement with in situ translocation studies (LaPointe et al., 2005; Yu & Haslam, 2005).
As protein synthesis inhibition was partially retained while VT1 transport to the Golgi was blocked at 16 °C or with BFA, alternative transport and translocation routes for VT1 may be possible. Under these conditions, high VT1 concentrations/longer incubation times are required to inhibit protein synthesis. These pathways to cytotoxicity are thus inefficient and are unlikely to be pathophysiologically significant, but demonstrate that the toxin is capable of exploiting multiple pathways to the cytosol and/or to inhibit protein synthesis. We did not detect any VTA1 cytosolic translocation with 16 °C or BFA treatments; however, it is possible that undetectable levels of VTA1 translocated. If VTA1 were able to translocate into the cytosol at 16 °C or with BFA treatment, protein synthesis inhibition should increase with time (Eiklid & Olsnes, 1980), as seen with BFA (Khine et al., 2004). In BFA-treated cells, VTA1 may be able to escape from the fused endosome/TGN compartment and translocate into the cytosol to inhibit protein synthesis. However, at 16 °C, protein synthesis inhibition remained steady at 50 % from 3 to 6 h of VT1 treatment and was not augmented by lactacystin proteosomal inhibition. This protein synthesis inhibition could result from signal transduction involving VTA or VT1 holotoxin, rather than from cytosolic translocation (Katagiri et al., 1999; Lauvrak et al., 2006; Mori et al., 2000), as VTB alone was incapable of reducing protein synthesis at 16 °C.
The intracellular transport of VT1 subunits is central to verotoxin-associated disease and to the use of verotoxin as an intracellular antigen-delivery agent. Both VT1 holotoxin and VTB can deliver antigens for MHC presentation, but how each targets antigens to the cytosol for presentation is unknown (Haicheur et al., 2003; Noakes et al., 1999; Vingert et al., 2006). Our results favour a retrograde ER translocation pathway in verotoxin-sensitive cells but a less efficient endosomal pathway for verotoxin-insensitive antigen presentation remains possible (Falguieres et al., 2001). The retrograde route used by VTA1 could target coupled antigen into the cytosol for processing and would explain why antigens attached to the VTA C terminus are not presented (Noakes et al., 1999), since they would be removed during activation. The intracellular transport routes used by VTB can vary between cell types (Falguieres et al., 2001); only lipid-raft-associated Gb3, dependent on Gb3 fatty acid composition (Falguieres et al., 2006), may mediate Golgi/ER retrograde transport leading to cytotoxicity. Gb3 raft association has been implicated in ER translocation of VTA (Smith et al., 2006) and ER retrograde transport of the cholera toxin B subunit is raft dependent (Fujinaga et al., 2003). However, unlike cholera toxin, VTA contains no ER-targeting motif (Jackson et al., 1999) and depends on VTB association for ER targeting, and hence translocation.
In our studies, we found no evidence of VTB translocation, but in non-raft Gb3-containing cells in which the lysosome is targeted (Falguieres et al., 2001; Hoey et al., 2003), this might occur (Falguieres & Johannes, 2006). The VT1 subunit transport in such cells, insensitive to VT1 protein synthesis inhibition and cytotoxicity, is a valid target for future work, since these cells can undergo VTA-dependent activation of selective mRNA translation (Harrison et al., 2005; Sakiri et al., 1998).
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ACKNOWLEDGEMENTS |
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Edited by: M. P. Stevens
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Received 19 February 2007;
revised 20 April 2007;
accepted 25 April 2007.
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) or dual-labelled VT1 (
). Mean; n=4. The experiment was repeated three times. CD50 values: VT, 3.5 pg ml–1; dual-labelled VT, 3.8 pg ml–1. Bioactivity was unaffected by labelling, separation and refolding of VTA and VTB.
