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Ludger Johannes

Publications and source records attributed to Ludger Johannes.

At least 19 recordsLinked to original sources
PubMed2006

Functionally different pools of Shiga toxin receptor, globotriaosyl ceramide, in HeLa cells.

Many studies have investigated the intracellular trafficking of Shiga toxin, but very little is known about the underlying dynamics of its cellular receptor, the glycosphingolipid globotriaosyl ceramide. In this study, we show that globotriaosyl ceramide is required not only for Shiga toxin binding to cells, but also for its intracellular trafficking. Shiga toxin induces globotriaosyl ceramide recruitment to detergent-resistant membranes, and subsequent internalization of the lipid. The globotriaosyl ceramide pool at the plasma membrane is then replenished from internal stores. Whereas endocytosis is not affected in the recovery condition, retrograde transport of Shiga toxin to the Golgi apparatus and the endoplasmic reticulum is strongly inhibited. This effect is specific, as cholera toxin trafficking on GM(1) and protein biosynthesis are not impaired. The differential behavior of both toxins is also paralleled by the selective loss of Shiga toxin association with detergent-resistant membranes in the recovery condition, and comparison of the molecular species composition of plasma membrane globotriaosyl ceramide indicates subtle changes in favor of unsaturated fatty acids. In conclusion, this study demonstrates the dynamic behavior of globotriaosyl ceramide at the plasma membrane and suggests that globotriaosyl ceramide-specific determinants, possibly its molecular species composition, are selectively required for efficient retrograde sorting on endosomes, but not for endocytosis.

Biological Transport, Active↗
PubMed2006

In vivo tumor targeting using a novel intestinal pathogen-based delivery approach.

Efficient methods for tumor targeting are eagerly awaited and must satisfy several challenges: molecular specificity, transport through physiologic barriers, and capacity to withstand extracellular or intracellular degradation and inactivation by the immune system. Through interaction with its hosts, the intestinal pathogen-produced Shiga toxin has evolved molecular properties that are of interest in this context. Its nontoxic B-subunit binds to the cellular toxin receptor, glycosphingolipid Gb3, which is highly expressed on human cancers and has recently been reported to be involved in the formation of metastasis in colorectal cancers. Its function as a target for cancer therapy has already been addressed in xenograft experiments. We here show that after oral or i.v. injections in mice, the B-subunit targets spontaneous digestive Gb3-expressing adenocarcinomas. The nontumoral mucosa is devoid of labeling, with the exception of rare enteroendocrine and CD11b-positive cells. As opposed to other delivery tools that are often degraded or recycled on cancer cells, the B-subunit stably associates with these cells due to its trafficking via the retrograde transport route. This can be exploited for the in vivo delivery of contrast agents to tumors, as exemplified using fibered confocal fluorescence endoscopy and positron emission tomography (PET) imaging. In conclusion, the data presented in this manuscript lay the groundwork for a novel delivery technology that, in addition to its use for molecular imaging applications such as noninvasive PET, could also be exploited for targeted tumor therapies.

Adenocarcinoma↗
PubMed2006

Synthesis of globo- and isoglobotriosides bearing a cinnamoylphenyl tag as novel electrophilic thiol-specific carbohydrate reagents.

The galactosyl donor, 4,6-di-O-acetyl-2,3-di-O-benzyl-D-galactopyranosyl trichloroacetimidate, was efficiently coupled with regioselectively benzylated lactoside acceptors under standard conditions to stereoselectively afford the corresponding globotrioside and isoglobotrioside derivatives in very good yields. These glycosides were smoothly functionalized with a 6-(p-cinnamoylphenoxy)-hexyl tether tag as novel electrophilic thiol-specific carbohydrate reagents. Immobilization of the globotrioside conjugate to Thiopropyl Sepharose 6B for purification of B-subunit of Shiga toxin (StxB) and coupling of a model cysteine-containing protein (StxB-Z(n)-Cys) to the isoglobotrioside conjugate were both performed with high efficiency.

Carbohydrate Sequence↗
PubMed2006

Characterization of the non-native trifluoroethanol-induced intermediate conformational state of the Shiga toxin B-subunit.

The effect of increasing concentrations of 2,2,2-trifluoroethanol (TFE) on the conformational stability of the Shiga toxin B-subunit (STxB), a bacterial homopentameric protein involved in cell-surface binding and intracellular transport, has been studied by far-, near-UV circular dichroism (CD), intrinsic fluorescence, analytical ultracentrifugation, and differential scanning calorimetry (DSC) under equilibrium conditions. Our data show that the native structure of STxB is highly perturbed by the presence of TFE. In fact, at concentrations of TFE above 20% (v/v), the native pentameric conformation of the protein is cooperatively transformed into a helix-rich monomeric and partially folded conformational state with no significant tertiary structure. Additionally, no cooperative transition was detected upon a further increase in the TFE concentration (above 40% (v/v)). The thermal stability of STxB was investigated at several different TFE concentrations using DSC and CD spectroscopy. Thermal transitions at TFE concentrations of up to 20% (v/v) were successfully fitted to the two-state folding/unfolding coupled to oligomerization model consistent with the transition between a pentameric folded conformation to a monomeric state of the protein, which the presence of TFE stabilizes as a partially folded conformation.

Calorimetry, Differential Scanning↗
PubMed2006

Stat-mediated signaling induced by type I and type II interferons (IFNs) is differentially controlled through lipid microdomain association and clathrin-dependent endocytosis of IFN receptors.

Type I (alpha/beta) and type II (gamma) interferons (IFNs) bind to distinct receptors, although they activate the same signal transducer and activator of transcription, Stat1, raising the question of how signal specificity is maintained. Here, we have characterized the sorting of IFN receptors (IFN-Rs) at the plasma membrane and the role it plays in IFN-dependent signaling and biological activities. We show that both IFN-alpha and IFN-gamma receptors are internalized by a classical clathrin- and dynamin-dependent endocytic pathway. Although inhibition of clathrin-dependent endocytosis blocked the uptake of IFN-alpha and IFN-gamma receptors, this inhibition only affected IFN-alpha-induced Stat1 and Stat2 signaling. Furthermore, the antiviral and antiproliferative activities induced by IFN-alpha but not IFN-gamma were also affected. Finally, we show that, unlike IFN-alpha receptors, activated IFN-gamma receptors rapidly become enriched in plasma membrane lipid microdomains. We conclude that IFN-R compartmentalization at the plasma membrane, through clathrin-dependent endocytosis and lipid-based microdomains, plays a critical role in the signaling and biological responses induced by IFNs and contributes to establishing specificity within the Jak/Stat signaling pathway.

Active Transport, Cell Nucleus↗
PubMed2006

The Shiga toxin B-subunit targets antigen in vivo to dendritic cells and elicits anti-tumor immunity.

The non-toxic B-subunit of Shiga toxin (STxB) interacts with the glycolipid Gb3, which is preferentially expressed on dendritic cells (DC) and B cells. After administration of STxB chemically coupled to OVA (STxB-OVA) in mice, we showed that the immunodominant OVA(257-264) peptide restricted by K(b) molecules is specifically presented by CD11c+ CD8alpha- DC, some of them displaying a mature phenotype. Using mice carrying a transgene encoding a diphtheria toxin receptor (DTR) under the control of the murine CD11c promoter, which allows inducible ablation of DC, we showed that DC are required for efficient priming of CTL after STxB-OVA vaccination. Immunization of mice with STxB-OVA induced OVA-specific CD8+ T cells detected ex vivo; these cells were long lasting, since they could be detected even 91 days after the last immunization and were composed of both central and memory T cells. Vaccination of mice with STxB-OVA and STxB coupled to E7, a protein derived from HPV16, inhibited tumor growth in prophylactic and therapeutic experiments. This effect was mainly mediated by CD8+ T cells. STxB therefore appears to be a powerful carrier directly targeting DC in vivo, resulting in a strong and durable CTL response associated with tumor protection.

Animals↗
PubMed2006

Shiga toxin B-subunit binds to the chaperone BiP and the nucleolar protein B23.

BACKGROUND INFORMATION: In many cell lines, such as HeLa cells, STxB (Shiga toxin B-subunit) is transported from the plasma membrane to the ER (endoplasmic reticulum), via early/recycling endosomes and the Golgi apparatus, bypassing the late endocytic pathway. In human monocyte-derived macrophages and dendritic cells that are not sensitive to Shiga toxin-induced protein biosynthesis inhibition, STxB is not detectably targeted to the retrograde route and is degraded in late endosomes/lysosomes. RESULTS: We have identified B-subunit interacting proteins in HeLa cells and macrophages. In HeLa cells, the ER-localized chaperone BiP (binding protein) was co-immunoprecipitated with the B-subunit. This interaction was not observed in macrophages, consistent with our previous trafficking results. In both cell types, the B-subunit also interacted with the nucleolar protein B23. Consistently, the B-subunit could be detected on nucleoli, suggesting that it could serve to bring the holotoxin to the site of synthesis of its molecular target, rRNA. The nucleolar localization data are critically discussed. CONCLUSION: The interaction of STxB with BiP, involved in the retrotranslocation process to the cytosol and nucleolar B23, as described in this study, might be of relevance for explaining the efficiency of even low doses of Shiga toxin to inactivate cellular ribosomes, and for the use of STxB as a vector for targeting antigens to cytosolic proteasomes of the MHC I-restricted antigen presentation pathway.

Active Transport, Cell Nucleus↗
PubMed2006

Internalized Pseudomonas exotoxin A can exploit multiple pathways to reach the endoplasmic reticulum.

Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the intoxication of mammalian cells by Pseudomonas exotoxin A (PEx). The toxin binds the alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein. Here, we show that in HeLa cells, PEx recruits a proportion of this receptor to detergent-resistant microdomains (DRMs). Uptake of receptor-bound PEx involves transport steps both directly from early endosomes to the trans-Golgi network (TGN) independently of Rab9 function and from late endosomes to the TGN in a Rab9-dependent manner. Furthermore, treatments that simultaneously perturb both Arf1-dependent and Rab6-dependent retrograde pathways show that PEx can use multiple routes to reach the ER. The Rab6-dependent route has only been described previously for cargo with lipid-sorting signals. These findings suggest that partial localization of PEx within DRM permits a choice of trafficking routes consistent with a model that DRM-associated toxins reach the ER on a lipid-dependent sorting pathway whilst non-DRM-associated PEx exploits the previously characterized KDEL receptor-mediated uptake pathway. Thus, unexpectedly, an ER-directed toxin with a proteinaceous receptor shows promiscuity in its intracellular trafficking pathways, exploiting routes controlled by both lipid- and protein-sorting signals.

3T3 Cells↗
PubMed2006

Rab6A and Rab6A' GTPases play non-overlapping roles in membrane trafficking.

The closely related Rab6 isoforms, Rab6A and Rab6A', have been shown to regulate vesicular trafficking within the Golgi and post-Golgi compartments, but studies using dominant active or negative mutant suggested conflicting models. Here, we report that reduction in the expression of Rab6 isoform using specific small interfering RNA reveals noticeable differences in the Rab6A and Rab6A' biological functions. Surprisingly, Rab6A seems to be largely dispensable in membrane trafficking events, whereas knocking down the expression of Rab6A' hampers the intracellular transport of the retrograde cargo marker, the Shiga Toxin B-subunit along the endocytic pathway, and causes defects in Golgi- associated protein recycling through the endoplasmic reticulum. We also showed that Rab6A' is required for cell cycle progression through mitosis and identify Ile(62) as a key residue for uncoupling Rab6A' functions in mitosis and retrograde trafficking. Thus, our work shows that Rab6A and Rab6A' perform different functions within the cell and suggests a novel role for Rab6A' as the major Rab6 isoform regulating previously described Rab6-dependent transport pathways.

Alternative Splicing↗
PubMed2005

The association of Shiga-like toxin with detergent-resistant membranes is modulated by glucosylceramide and is an essential requirement in the endoplasmic reticulum for a cytotoxic effect.

Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the productive intoxication of susceptible mammalian cells by Shiga-like toxin-1 (SLTx). Recently, it has been proposed that the observed association of certain ER-directed toxins and viruses with detergent-resistant membranes (DRM) may provide a general mechanism for their retrograde transport to endoplasmic reticulum (ER). Here, we show that DRM recruitment of SLTx bound to its globotriosylceramide (Gb(3)) receptor is mediated by the availability of other glycosphingolipids. Reduction in glucosylceramide (GlcCer) levels led to complete protection against SLTx and a reduced cell surface association of bound toxin with DRM. This reduction still allowed efficient binding and transport of the toxin to the ER. However, toxin sequestration within DRM of the ER was abolished under reduced GlcCer conditions, suggesting that an association of toxin with lipid microdomains or rafts in the ER (where these are defined by detergent insolubility) is essential for a later step leading to or involving retro-translocation of SLTx across the ER membrane. In support of this, we show that a number of ER residents, proteins intimately involved in the process of ER dislocation of misfolded proteins, are present in DRM.

1-Deoxynojirimycin↗
PubMed2005

Lowe syndrome protein OCRL1 interacts with clathrin and regulates protein trafficking between endosomes and the trans-Golgi network.

Oculocerebrorenal syndrome of Lowe is caused by mutation of OCRL1, a phosphatidylinositol 4,5-bisphosphate 5-phosphatase localized at the Golgi apparatus. The cellular role of OCRL1 is unknown, and consequently the mechanism by which loss of OCRL1 function leads to disease is ill defined. Here, we show that OCRL1 is associated with clathrin-coated transport intermediates operating between the trans-Golgi network (TGN) and endosomes. OCRL1 interacts directly with clathrin heavy chain and promotes clathrin assembly in vitro. Interaction with clathrin is not, however, required for membrane association of OCRL1. Overexpression of OCRL1 results in redistribution of clathrin and the cation-independent mannose 6-phosphate receptor (CI-MPR) to enlarged endosomal structures that are defective in retrograde trafficking to the TGN. Depletion of cellular OCRL1 also causes partial redistribution of a CI-MPR reporter to early endosomes. These findings suggest a role for OCRL1 in clathrin-mediated trafficking of proteins from endosomes to the TGN and that defects in this pathway might contribute to the Lowe syndrome phenotype.

Animals↗
PubMed2005

tGolgin-1 (p230, golgin-245) modulates Shiga-toxin transport to the Golgi and Golgi motility towards the microtubule-organizing centre.

tGolgin-1 (trans-Golgi p230, golgin-245) is a member of a family of large peripheral membrane proteins that associate with the trans-Golgi network (TGN) via a C-terminal GRIP domain. Some GRIP-domain proteins have been implicated in endosome-to-TGN transport but no function for tGolgin-1 has been described. Here, we show that tGolgin-1 production is required for efficient retrograde distribution of Shiga toxin from endosomes to the Golgi. Surprisingly, we also found an indirect requirement for tGolgin-1 in Golgi positioning. In HeLa cells depleted of tGolgin-1, the normally centralized Golgi and TGN membranes were displaced to the periphery, forming 'mini stacks'. These stacks resembled those in cells with disrupted microtubules or dynein-dynactin motor, in that they localized to endoplasmic-reticulum exit sites, maintained their secretory capacity and cis-trans polarity, and were relatively immobile by video microscopy. The mini stacks formed concomitant with a failure of pre-Golgi elements to migrate along microtubules towards the microtubule-organizing centre. The requirement for tGolgin-1 in Golgi positioning did not appear to reflect direct binding of tGolgin-1 to motile pre-Golgi membranes, because distinct Golgi and tGolgin-1-containing TGN elements that formed after recovery of HeLa cells from brefeldin-A treatment moved independently toward the microtubule-organizing centre. These data demonstrate that tGolgin-1 functions in Golgi positioning indirectly, probably by regulating retrograde movement of cargo required for recruitment or activation of dynein-dynactin complexes on newly formed Golgi elements.

Autoantigens↗
PubMed2005

Cholera and Shiga toxin B-subunits: thermodynamic and structural considerations for function and biomedical applications.

The B-subunits of cholera and Shiga toxins are functionally and structurally related proteins with different chain lengths and no sequence similarity. They are responsible for toxin binding to specific glycosphingolipid receptors and intracellular toxin trafficking. Indeed, it is clearly established that B-subunits have the unique capacity of targeting the toxins to a poorly explored intracellular pathway, the retrograde route, allowing the transfer to the cytosol of the associated catalytic A-subunits, by retro-translocation from the endoplasmic reticulum. The B-subunits have also been used as vectors for antigen presentation in immunotherapeutic approaches. It is, however, not known if and how the B-subunits intervene in membrane translocation of the A-subunits and/or antigens to the cytosol. Therefore, it is important to characterise the driving force of pentamer formation, its conformational stability, and toxin-receptor interactions. This review summarises recent studies that have dealt with these topics.

Amino Acid Sequence↗
PubMed2005

Protein interaction mapping: a Drosophila case study.

The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (http://pim.hybrigenics.com), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps.

Animals↗
PubMed2005

Functional analysis of Arl1 and golgin-97 in endosome-to-TGN transport using recombinant Shiga toxin B fragment.

A direct transport route from early/recycling endosome (EE/RE) to the trans-Golgi network (TGN) is exploited by Shiga toxin (Mallard et al., 1998) and TGN38 (Ghosh et al., 1998). To facilitate the study of this pathway, both in vivo and in vitro transport assays using recombinant Shiga toxin B fragments (STxB) as protein cargos have facilitated the analysis of this transport event (Johannes et al., 1997; Mallard et al., 1998, 2002; Tai et al., 2004). We describe here the application of these assays to study the role of a small GTPase Arl1 and its effector golgin-97 in this transport process.

ADP-Ribosylation Factors↗
PubMed2004

Participation of the syntaxin 5/Ykt6/GS28/GS15 SNARE complex in transport from the early/recycling endosome to the trans-Golgi network.

An in vitro transport assay, established with a modified Shiga toxin B subunit (STxB) as a marker, has proved to be useful for the study of transport from the early/recycling endosome (EE/RE) to the trans-Golgi network (TGN). Here, we modified this assay to test antibodies to all known soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that have been shown to localize in the Golgi and found that syntaxin 5, GS28, Ykt6, and GS15 antibodies specifically inhibited STxB transport. Because syntaxin 5, GS28, Ykt6, and GS15 exist as a unique SNARE complex, our observation indicates that these four SNAREs function as a complex in EE/RE-TGN transport. The importance of GS15 in EE/RE-TGN transport was further demonstrated by a block in recombinant STxB transport in HeLa cells when GS15 expression was knocked down by its small interfering iRNA. Morphological analyses showed that some GS15 and Ykt6 were redistributed from the Golgi to the endosomes when the recycling endosome was perturbed by SNX3-overexpression, suggesting that GS15 and Ykt6 might cycle between the endosomes and the Golgi apparatus. Further studies indicated that syntaxin 5 and syntaxin 16 exerted their role in EE/RE-TGN transport in an additive manner. The kinetics of inhibition exhibited by syntaxin 16 and syntaxin 5 antibodies is similar.

Animals↗
PubMed2004

Glycosphingolipids as toxin receptors.

A number of proteins produced by certain bacteria and plants are potently toxic to mammalian cells. This toxicity results from their ability to catalytically modify macromolecules that are required for essential cellular functions such as vesicular trafficking, cytoskeletal assembly, signalling or protein synthesis. To reach their targets, these proteins bind specific surface receptors before endocytosis and translocation across an internal membrane. The surface receptors exploited by different toxins include a range of proteins and lipids. Here we focus on specific glycosphingolipid receptors and two well-characterised subsets of toxins that exploit them for surface binding, intracellular trafficking, and signalling events.

Animals↗