Effect of Globotriaosyl Ceramide Fatty Acid α-Hydroxylation on the Binding by Verotoxin 1 and Verotoxin 2

Variation in the lipid moiety of the verotoxin (VT) receptor glycosphingolipid, globotriaosyl ceramide (Gb₃) can modulate toxin binding. The binding of VT1 and VT2 to C18 and C22 ahydroxy and nonhydroxy fatty acid isoforms of Gb₃ were compared using a receptor ELISA and a ¹²⁵l-labeled toxin/glycolipid microtitre plate direct binding assay. Increased binding to the hydroxylated species, particularly C22OH, was observed for both toxins. Increased RELISA binding at low glycolipid concentrations only, suggested the binding affinity is increased following Gb₃ fatty acid hydroxylation. Nonlinear regression analysis of direct binding assay to these Gb₃ isoforms confirmed the increased affinity of both toxins for the C22 hydroxylated Gb₃. The capacity was also significantly increased. The increased binding of VTs for hydroxylated fatty acid Gb₃ isoforms may be a factor in the selective renal pathology which can follow systemic verotoxemia, particularly in the mouse model. The more pronounced effect at lower glycolipid concentrations prompted investigation of VT1 binding affinity at different Gb₃ concentrations. Unexpectedly, the VT1 Kd for Gb₃ was found to decrease as an inverse function of the Gb₃ concentration. This shows that glycolipids have nonclassical receptor properties.     

Capping and receptor-mediated endocytosis of cell-bound verotoxin (shiga-like toxin) 1: Chemical identification of an amino acid in the B subunit necessary for efficient receptor glycolipid binding and cellular internalization

The glycolipid globotriaosylceramide (Gb₃) is the plasma membrane receptor that mediates the internalization of verotoxin (VT1) into susceptible cells by capping and receptor-mediated endocytosis (RME). Internalization of fluorescein isothiocyanate-conjugated holotoxin into Daudi lymphoma cells was found to be slower than the pentameric receptor binding B subunit alone, suggesting that the A subunit may interact with the membrane to compromise the lateral mobility of the receptor bound B subunit. 3-D reconstruction of fluorescent images by confocal microscopy confirmed the complete internalization of holotoxin. VT1 internalization and cytotoxicity was inhibited by monodansyl cadavarine, which supports a role for clathrin coated pits in the RME of VT1. Biotinylation of the B subunit (in contrast to fluorescein labelling) was found to prevent toxin internalization. This effect correlated with reduced binding of Gb₃ and reduced cytotoxicity in vitro. By cleavage of the B subunit at the single tryptophan residue, the reduced Gb₃ binding and lack of cellular internalization was shown to be due to the biotinylation of lysine 53 in the VT1 B subunit. This residue was not labelled with fluorescein isothiocyanate in the native protein. This conclusion was confirmed by the finding that biotinylation of VT2c (which contains lys 53) prevented glycolipid receptor binding, whereas biotinylation of VT2e (in which lys 53 is substituted by ile) had no effect. © 1994 Wiley-Liss, Inc.     

Globotriosyl ceramide is specifically recognized by the Escherichia coli verocytotoxin 2

Two Escherichia coli cytotoxins (verotoxins 1 and 2) have been previously implicated in the cytopathology of the Hemolytic Uremic Syndrome. We have examined the glycolipid binding specificity of verotoxin (VT)2. This toxin specifically binds to globotriosyl ceramide (galactose alpha 1-4 galactose beta 1-4 glucosyl ceramide). Removal, or substitution of the terminal a galactose residue with N-acetyl galactosamine in beta 1-3 linkage, deletes binding activity. The toxin does not recognize similar terminal a galactose residues on a glycoglycerolipid. Thus the binding specificity of VT2 is the same as previously reported for VT1. Liposomes containing globotriosyl ceramide are able to specifically remove VT1 and VT2 cytotoxicity and cell lines selected in vitro for resistance to VT1 are cross resistant to VT2.     

Intracellular targeting of the endoplasmic reticulum/nuclear envelope by retrograde transport may determine cell hypersensitivity to verotoxin via globotriaosyl ceramide fatty acid isoform traffic

The pentameric B subunit of verotoxin (VT) mediates the attachment to cell surface globotriaosyl ceramide (Gb3) to facilitate receptor-mediated endocytosis of the toxin. In highly toxin-sensitive tumor cells, the holotoxin and VT1 B subunit is targeted intracellularly to elements of the endoplasmic reticulum (ER)/nuclear membrane. In less sensitive cells, the toxin is targeted to components of the Golgi apparatus. We have studied two cell systems: the induced VT hypersensitivity of human astrocytoma cell lines cultured in the presence of sodium butyrate (compared to sodium propionate and capronate) and the increased VT sensitivity of multiple drug-resistant mutants as compared to parental human ovarian carcinoma cells. In both cases, a difference in the intracellular retrograde transport of the receptor-bound internalized toxin to the ER/nuclear envelope, as opposed to the Golgi, correlated with a >1,000-fold increase in cell sensitivity to VT. This change in intracellular routing may be due to sorting of Gb3 fatty acid isoforms, since nuclear targeting was found in turn to correlate with the preferential synthesis of Gb3 containing shorter chain (primarily C16) fatty acid species. We propose that the isoform-dependent traffic of Gb3 from the cell surface to the ER/nuclear membrane provides a new signal transduction pathway for Gb3 binding proteins.     

Influence of phospholipid chain length on verotoxin/globotriaosyl ceramide binding in model membranes: comparison of a supported bilayer film and liposomes

The importance of the surrounding lipid environment on the availability of glycolipid carbohydrate for ligand binding was demonstrated by studying the influence of phosphatidylcholine fatty acid chain length on binding of verotoxins (VT1 and VT2c) to their specific cell surface receptor, globotriaosylceramide (Gb₃) in the presence of auxiliary lipids both in a microtitre plate surface bilayer film and in a liposome membrane model system. In the microtitre assay, both VT1 and VT2c binding to Gb₃ was increased as a function of decreasing PC acyl chain length likely resulting in increased Gb₃ exposure. In the liposome assay VT1 binding was similarly modulated, however the effect on VT2c binding was more complex and did not follow a simple function of increased carbohydrate exposure. Earlier work established that C22:1 and C18:1Gb₃ fatty acid homologues were the preferred Gb₃ receptor isoforms in the microtitre assay for VT1 and VT2c respectively. This selectivity was maintained in C16PC containing liposomes, but in C14PC liposomes, binding to C22:1Gb₃ (but not C18:1Gb₃) was elevated such that this Gb₃ species now became the preferred receptor for both toxins. This change in verotoxin/Gb₃ homologue binding selectivity in the presence of C14PC did not occur in the microtitre bilayer format. These results are consistent with our proposal that these toxins recognize different epitopes on the Gb₃ oligosaccharide. We infer that relative availability of these epitopes for toxin binding in an artificial bilayer is influenced not only by the exposure due to the discrepancy between the fatty acyl chain lengths of Gb₃ and PC, but by the physical mode of presentation of the bilayer structure. Such acyl chain length differences have a more marked effect in a supported bilayer film whereas only the largest discrepancies affect Gb₃ receptor function in liposomes. The basis of phospholipid modulation of glycolipid carbohydrate accessibility for receptor function is likely complex and will involve phase separation, gel/liquid crystalline transition, packing and lateral mobility within the bilayer, suggesting that such parameters should be considered in the assessment of glycolipid receptor function in cells.     

Glycolipid receptors for verotoxin and Helicobacter pylori: role in pathology

Eukaryotic cell surface glycolipids can act as both the primary interface between bacteria and their host and secondly as a targeting mechanism for bacterial virulence factors. The former is characterized by redundancy in adhesin-receptor interactions and the latter by a higher affinity, more restrictive glycolipid binding specificity for targeting. Interactions of verotoxin with its glycolipid receptor globotriaosylceramide and Helicobacter pylori binding to a variety of different glycolipids, which can be environmentally regulated, provide examples of these differing modes of glycolipid receptor function. Verotoxins are involved in endothelial targeting in the microangiopathies of hemorrhagic colitis and hemolytic uremic syndrome (HUS). The highly restricted binding specificity and crystal structure of the verotoxin B subunit have allowed theoretical modeling of the Gb3 binding site of the verotoxin B subunit pentamer which provides an approach to intervention. Studies of the role of glycolipid function in verotoxin-induced disease have concentrated on the distribution of Gb3 and its ability to mediate the internalization of the toxin within the target cell. The distribution of Gb3 within the renal glomerulus plays a central role in defining the age-related etiology of HUS following gastrointestinal infection with VT producing Escherichia coli. H. pylori, on the other hand, instigates a less distinct but more complex disseminated gastric inflammation. Studies on the role of glycolipid receptors in H. pylori infection have been bogged down in establishing the importance of each binding specificity defined. In addition, the physiological condition of the organism within the various binding assays has not been extensively considered, such that spurious non-physiological interactions may have been elucidated. The identification and cloning of a Le(b) binding adhesin and the identification of cell surface hsp70 as a mediator of sulfoglycolipid binding under stress conditions may now allow a more molecular approach to define the role of glycolipid recognition in this infection.     

Retroviral transfection of Madin-Darby canine kidney cells with human MDR1 results in a major increase in globotriaosylceramide and 10(5)- to 10(6)-fold increased cell sensitivity to verocytotoxin. Role of p-glycoprotein in glycolipid synthesis

Retroviral infection of the Madin-Darby canine kidney (MDCK) renal cell line with human MDR1 cDNA, encoding the P-glycoprotein (P-gp) multidrug resistance efflux pump, induces a major accumulation of the glycosphingolipid (GSL), globotriaosylceramide (Galalpha1-4Galbeta1-4glucosylceramide-Gb(3)), the receptor for the E. coli-derived verotoxin (VT), to effect a approximately million-fold increase in cell sensitivity to VT. The shorter chain fatty acid isoforms of Gb(3) (primarily C16 and C18) are elevated and VT is internalized to the endoplasmic reticulum/nuclear envelope as we have reported for other hypersensitive cell lines. P-gp (but not MRP) inhibitors, e.g. ketoconazole or cyclosporin A (CsA) prevented the increased Gb(3) and VT sensitivity, concomitant with increased vinblastine sensitivity. Gb(3) synthase was not significantly elevated in MDR1-MDCK cells and was not affected by CsA. In MDR1-MDCK cells, synthesis of fluorescent N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-aminocaproyl (NBD)-lactosylceramide (LacCer) and NBD-Gb(3) via NBD-glucosylceramide (GlcCer) from exogenous NBD-C(6)-ceramide, was prevented by CsA. We therefore propose that P-gp can mediate GlcCer translocation across the bilayer, from the cytosolic face of the Golgi to the lumen, to provide increased substrate for the lumenal synthesis of LacCer and subsequently Gb(3). These results provide a molecular mechanism for the observed increased sensitivity of multidrug-resistant tumors to VT and emphasize the potential of verotoxin as an antineoplastic. Two strains (I and II) of MDCK cells, which differ in their glycolipid profile, have been described. The original MDR1-MDCK parental cell was not specified, but the MDR1-MDCK GSL phenotype and glycolipid synthase activities indicate MDCK-I cells. However, the partial drug resistance of MDCK-I cells precludes their being the parental cell. We speculate that the retroviral transfection per se, or the subsequent selection for drug resistance, selected a subpopulation of MDCK-I cells in the parental MDCK-II cell culture and that drug resistance in MDR1-MDCK cells is thus a result of both MDR1 expression and a second, previously unrecognized, component, likely the high level of GlcCer synthesis in these cells.     

Fusion Toxin BLyS-Gelonin Inhibits Growth of Malignant Human B Cell Lines In Vitro and In Vivo

B lymphocyte stimulator (BLyS) is a member of the TNF superfamily of cytokines. The biological activity of BLyS is mediated by three cell surface receptors: BR3/BAFF-R, TACI and BCMA. The expression of these receptors is highly restricted to B cells, both normal and malignant. A BLyS-gelonin fusion toxin (BLyS-gel) was generated consisting of the recombinant plant-derived toxin gelonin fused to the N-terminus of BLyS and tested against a large and diverse panel of B-NHL cell lines. Interestingly, B-NHL subtypes mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL) and B cell precursor-acute lymphocytic leukemia (BCP-ALL) were preferentially sensitive to BLyS-gel mediated cytotoxicity, with low picomolar EC₅₀ values. BLyS receptor expression did not guarantee sensitivity to BLyS-gel, even though the construct was internalized by both sensitive and resistant cells. Resistance to BLyS-gel could be overcome by treatment with the endosomotropic drug chloroquine, suggesting BLyS-gel may become trapped within endosomal/lysosomal compartments in resistant cells. BLyS-gel induced cell death was caspase-independent and shown to be at least partially mediated by the “ribotoxic stress response.” This response involves activation of p38 MAPK and JNK/SAPK, and BLyS-gel mediated cytotoxicity was inhibited by the p38/JNK inhibitor SB203580. Finally, BLyS-gel treatment was shown to localize to sites of disease, rapidly reduce tumor burden, and significantly prolong survival in xenograft mouse models of disseminated BCP-ALL, DLBCL, and MCL. Together, these findings suggest BLyS has significant potential as a targeting ligand for the delivery of cytotoxic “payloads” to malignant B cells.     

Aglycone modulation of glycolipid receptor function

The aglycone has been largely ignored in consideration of glycoconjugate function. Evidence is reviewed which suggests that the role of the lipid in glycolipid carbohydrate function may be particularly significant. The lipid moiety can promote or reduce carbohydrate exposure of membrane glycolipids. Theoretical calculation has indicated that the plane of the plasma membrane can restrict the permitted conformations of a given glycolipid oligosaccharide. Thus the lipid moiety may influence the relative conformation of such carbohydrate sequences. Evidence of ceramide regulation of glycolipid function can be found in studies of enzyme substrate specificity, antiglycolipid recognition and bacterial/host cell interactions. Studies of verotoxin binding to its glycolipid receptor globotriaosyl ceramide indicate that modulation of receptor function by glycolipid fatty acid content plays an important role inin vitro binding assays, cell cytotoxicity and intracellular routing.     

Glycolipid binding of purified and recombinant Escherichia coli produced verotoxin in vitro

Escherichia coli verotoxin (also known as Shiga-like toxin) has been implicated in the aetiology of the hemolytic uremic syndrome and hemorrhagic colitis. The glycolipid binding specificity of verotoxin purified from E. coli H30 and verotoxin cloned from bacteriophage H19B has been examined. Verotoxin from both sources binds specifically to globotriosyl ceramide containing the carbohydrate sequence galactose alpha 1-4galactose beta 1-4glucose-ceramide. Removal of the terminal galactose or substitution with N-acetylgalactosamine in beta 1-3 linkage deletes toxin binding activity. A ceramide trihexoside species, consistent with a globotriosyl ceramide structure was shown to be the major verotoxin-binding glycolipid of cultured vero cells which are routinely used to measure the cytotoxicity of toxin samples.     

Functional significance of globotriaosyl ceramide in interferon-alpha(2)/type 1 interferon receptor-mediated antiviral activity

The N-terminus of the type 1 interferon receptor subunit, IFNAR1, has high amino acid sequence similarity to the receptor binding B subunit of the Escherichia coli-derived verotoxin 1, VT1. The glycolipid, globotriaosyl ceramide (Gb(3): Gal alpha(1) --> 4 Gal beta 1 --> 4 Glu beta 1 --> 1 Cer) is the specific cell receptor for VT1. Gb(3)-deficient variant cells selected for VT resistance are cross-resistant to interferon-alpha (IFN-alpha)-mediated antiproliferative activity. The association of eIFNAR1 with Gal alpha 1 --> 4 Gal containing glycolipids has been previously shown to be important for the receptor-mediated IFN-alpha signal transduction for growth inhibition. The crucial role of Gb(3) for the signal transduction of IFN-alpha-mediated antiviral activity is now reported. IFN-alpha-mediated antiviral activity, nuclear translocation of activated Stat1, and increased expression of PKR were defective in Gb(3)-deficient vero mutant cells, although the surface expression of IFNAR1 was unaltered. The VT1B subunit was found to inhibit IFN-alpha-mediated antiviral activity, Stat1 nuclear translocation and PKR upregulation. Unlike VT1 cytotoxicity, IFN-alpha-induced Stat1 nuclear translocation was not inhibited when RME was prevented, suggesting that the accessory function of Gb(3) occurs at the plasma membrane. IFN-alpha antiviral activity was also studied in Gb(3)-positive MRC-5 cells, which are resistant to IFN-alpha growth inhibition, partially resistant to VT1 but still remain fully sensitive to IFN-alpha antiviral activity, and two astrocytoma cell lines expressing different Gb(3) fatty acid isoforms. In both systems, long chain fatty acid-containing Gb(3) isoforms, which are less effective to mediate VT1 cytotoxicity, were found to correlate with higher IFN-alpha-mediated antiviral activity. Inhibition of Gb(3) synthesis in toto prevented IFN-alpha antiviral activity in all cells. We propose that the long chain Gb(3) fatty isoforms preferentially remain in the plasma membrane, and by associating with IFNAR1, mediate IFN-alpha antiviral signaling, whereas short chain Gb(3) fatty acid isoforms are preferentially internalized to mediate VT1 cytotoxicity and IFNAR1-dependent IFN-alpha growth inhibition.     

A mutational analysis of the globotriaosylceramide-binding sites of verotoxin VT1.

Escherichia coli verotoxin, also known as Shiga-like toxin, binds to eukaryotic cell membranes via the glycolipid Gb(3) receptors which present the P(k) trisaccharide Galalpha(1-4)Galbeta(1-4)Glcbeta. Crystallographic studies have identified three P(k) trisaccharide (P(k)-glycoside) binding sites per verotoxin 1B subunit (VT1B) monomer while NMR studies have identified binding of P(k)-glycoside only at site 2. To understand the basis for this difference, we studied binding of wild type VT1B and VT1B mutants, defective at one or more of the three sites, to P(k)-glycoside and pentavalent P(k) trisaccharide (pentaSTARFISH) in solution and Gb(3) presented on liposomal membranes using surface plasmon resonance. Site 2 was the key site in terms of free trisaccharide binding since mutants altered at sites 1 and 3 bound this ligand with wild type affinity. However, effective binding of the pentaSTARFISH molecule also required a functional site 3, suggesting that site 3 promotes pentavalent binding of linked trisaccharides at site 1 and site 2. Optimal binding to membrane-associated Gb(3) involved all three sites. Binding of all single site mutants to liposomal Gb(3) was weaker than wild type VT1B binding. Site 3 mutants behaved as if they had reduced ability to enter into high avidity interactions with Gb(3) in the membrane context. Double mutants at site 1/site 3 and site 2/site 3 were completely inactive in terms of binding to liposomal Gb(3,) even though the site 1/site 3 mutant bound trisaccharide with almost wild type affinity. Thus site 2 alone is not sufficient to confer high avidity binding to membrane-localized Gb(3). Cytotoxic activity paralleled membrane glycolipid binding. Our data show that the interaction of verotoxin with the Gb(3) trisaccharide is highly context dependent and that a membrane environment is required for biologically relevant studies of the interaction.     

Verotoxin-resistant cell clones are deficient in the glycolipid globotriosylceramide: differential basis of phenotype

Escherichia coli-derived verotoxin is an extremely toxic protein and is highly selective toward certain primate cells. Two susceptible cell lines are the Daudi cell line (human Burkitt lymphoma) and the Vero cell line (Green African monkey kidney). Both of these cell lines contain significant levels of the verotoxin binding glycolipid globotriosylceramide (Gb3) (1 nmol/10(7) cells and 3 nmol/10(6) cells, respectively). A clone was selected from the Vero cell line for resistance to Verotoxin 2, while a mutant from the Daudi cell line was selected for resistance to Verotoxin 1. Both were found to be deficient in globotriosylceramide with a corresponding increase in the precursor glycolipid lactosylceramide. Cell free assay of alpha-galactosyltransferase activity revealed that the Vero cell clone (VRP) contained significantly reduced enzyme activity, whereas in the case of the Daudi mutant (VT20), no significant decrease in activity was noted in vitro. These observations suggest a complex regulation of Gb3 biosynthesis which is considered in relation to P blood group antigen expression.     

Synergistic effect of verotoxin and interferon-alpha on erythropoiesis.

Previous studies have shown that the verotoxin receptor globotriaosyl ceramide is involved in interferon-alpha (IFN-alpha) signaling pathways. The results of the present study indicate that verotoxin used in combination with IFN-alpha had a direct cytotoxic effect on erythrocyte development by targeting nucleated erythrocyte precursors. Toxin treatment alone had no significant effect on erythropoiesis. However, treatment with 100 ng/ml of verotoxin (VT) in combination with 100 U/ml of IFN-alpha was highly cytotoxic (>90%) to erythroid cells in human cord blood cultures by day 14 post-treatment. The lack of effect on other hematopoietic cells, and the relatively modest decrease in the number of erythroid colonies formed (28%) as a result of IFN-alpha/VT treatment, indicate that the cytotoxicity was targeted specifically toward cells committed to the erythrocyte lineage. IFN-alpha treatment alone did not result in cytotoxicity or a significant reduction in the number of erythroid colonies formed. However, IFN-alpha treatment did result in an increase in the surface expression of verotoxin receptors as determined by flow cytometry following labeling of cells with VT-FITC. Abnormalities in erythrocyte morphology and anemia are associated with infection by verotoxin-producing Escherichia coli such as serotype O157:H7. These symptoms are frequently attributed to passage of erythrocytes through partially occluded blood vessels following toxin-induced damage to endothelial cells. The present results document a synergistic cytotoxic effect of IFN-alpha and verotoxin on erythropoiesis, which could have relevance to clinical infection with verotoxin-producing bacteria.     

Mutant with diphtheria toxin receptor and acidification function but defective in entry of toxin

A mutant of Chinese hamster ovary cells, GE1, that is highly resistant to diphtheria toxin was isolated. The mutant contains 50% ADP-ribosylatable elongation factor 2, but its protein synthesis was not inhibited by the toxin even at concentrations above 100 μg/ml. ¹²⁵I-labeled diphtheria toxin was associated with GE1 cells as well as with the parent cells but did not block protein synthesis of GE1 cells even when the cells were exposed to low pH in the presence or absence of NH₄Cl. The infections of GE1 cells and the parent cells by vesicular stomatitis virus were similar. GE1 cells were cross-resistant to Pseudomonas aeruginosa exotoxin A and so were about 1000 times more resistant to this toxin than the parent cells. Hybrids of GE1 cells and the parent cells or mutant cells lacking a functional receptor were more sensitive to diphtheria toxin than GE1 cells. These results suggest that entry of diphtheria toxin into cells requires a cellular factor(s) in addition to those involved in receptor function and acidification of endosomes and that GE1 cells do not express this cellular factor. This character is recessive in GE1 cells.     

Escherichia coli verotoxin 1 mediates apoptosis in human HCT116 colon cancer cells by inducing overexpression of the GADD family of genes and S phase arrest

The Escherichia coli verotoxin 1 (VT1) inhibits protein synthesis, cell proliferation, and damages endothelial cell in the hemolytic uremic syndrome. VT1 can specifically bind and act on endothelial cells as well as on many tumor cells because these cells express its high affinity receptor, globotriaosylceramide. This indicates that VT1 may have both antiangiogenic and antineoplastic activities. We investigated this potential of VT1 by incubating several colon cancer cell lines with VT1 for different time periods and found that HCT116 cells were especially sensitive to VT1. A combination of morphological studies, flow cytometry, DNA laddering and annexin V staining confirmed that VT1 irreversibly arrests these cells in S phase within 24 h and prolonged incubation triggers DNA fragmentation. Concomitant to the activation of the S phase checkpoint, increased levels of mRNA and proteins of growth arrest and DNA damage-inducible gene family that include GADD34, GADD45alpha, and GADD45beta was observed. Interestingly, no significant changes in expression of key cell cycle related proteins such as cdk2, cdk4, p21, p27, and p53 was found during the S phase arrest and apoptosis. We therefore suggest that GADD proteins might play an important role in VT1 induced S phase arrest and programmed cell death in HCT116 cells.     

pH-independent retrograde targeting of glycolipids to the Golgi complex

A small fractionof the molecules internalized by endocytosis reaches the Golgi complexthrough a retrograde pathway that is poorly understood. In the presentwork, we used bacterial toxins to study the retrograde pathway in Verocells. The recombinant B subunit of verotoxin 1B (VT1B)was labeled with fluorescein to monitor its progresswithin the cell by confocal microscopy. This toxin, which bindsspecifically to the glycolipid globotriaosyl ceramide, enteredendosomes by both clathrin-dependent and -independent pathways,reaching the Golgi complex. Once internalized, the toxin-receptor complex did not recycle back to the plasma membrane. The kinetics ofinternalization and the subcellular distribution of VT1B were virtuallyidentical to those of another glycolipid-binding toxin, the B subunitof cholera toxin (CTB). Retrograde transport of VT1B and CTB wasunaffected by addition of weak bases in combination with concanamycin,a vacuolar-type ATPase inhibitor. Ratio imaging confirmed that theseagents neutralized the luminal pH of the compartments where the toxinwas located. Therefore, the retrograde transport of glycolipids differsfrom that of proteins like furin and TGN38, which require an acidicluminal pH. Additional experiments indicated that the glycolipidreceptors of VT1B and CTB are internalized independently and not aspart of lipid "rafts" and that internalization is cytochalasininsensitive. We conclude that glycolipids utilize a unique,pH-independent retrograde pathway to reach compartments of thesecretory system and that assembly of F-actin is not required for thisprocess.

     

Structure-dependent pseudoreceptor intracellular traffic of adamantyl globotriaosyl ceramide mimics

The verotoxin (VT) (Shiga toxin) receptor globotriaosyl ceramide (Gb(3)), mediates VT1/VT2 retrograde transport to the endoplasmic reticulum (ER) for cytosolic A subunit access to inhibit protein synthesis. Adamantyl Gb(3) is an amphipathic competitive inhibitor of VT1/VT2 Gb(3) binding. However, Gb(3)-negative VT-resistant CHO/Jurkat cells incorporate adaGb(3) to become VT1/VT2-sensitive. CarboxyadaGb(3), urea-adaGb(3), and hydroxyethyl adaGb(3), preferentially bound by VT2, also mediate VT1/VT2 cytotoxicity. VT1/VT2 internalize to early endosomes but not to Golgi/ER. AdabisGb(3) (two deacyl Gb(3)s linked to adamantane) protects against VT1/VT2 more effectively than adaGb(3) without incorporating into Gb(3)-negative cells. AdaGb(3) (but not hydroxyethyl adaGb(3)) incorporation into Gb(3)-positive Vero cells rendered punctate cell surface VT1/VT2 binding uniform and subverted subsequent Gb(3)-dependent retrograde transport to Golgi/ER to render cytotoxicity (reduced for VT1 but not VT2) brefeldin A-resistant. VT2-induced vacuolation was maintained in adaGb(3)-treated Vero cells, but vacuolar membrane VT2 was lost. AdaGb(3) destabilized membrane cholesterol and reduced Gb(3) cholesterol stabilization in phospholipid liposomes. Cholera toxin GM1-mediated Golgi/ER targeting was unaffected by adaGb(3). We demonstrate the novel, lipid-dependent, pseudoreceptor function of Gb(3) mimics and their structure-dependent modulation of endogenous intracellular Gb(3) vesicular traffic.     

Mechanism of resistance to ricin toxin in selected mouse lymphoma cell lines

The role of impaired toxin uptake in conferring cellular resistance to the plant toxin RCA_II (ricin) has been examined using a murine BW5 147 lymphoma line and a toxin-resistant variant (BW5 147 Ric^R.3) selected by repeated exposure to RCA_II. The toxin-resistant variant is 250 times more resistant to RCA_II in long-term growth experiments and 1,000 times more resistant in short-term protein synthesis assays. Experiments with ferritin-conjugated ¹²⁵I-labeled RCA_II (ferritin-¹²⁵I-RCA_II) indicated that toxin binding to sensitive and resistant cells is similar at low toxin concentrations where maximum differential cytotoxicity occurs but that major difference exist with respect to toxin uptake. In sensitive cells toxin is internalized via endocytosis, and as seen previously in other systems subsequent rupture of some of the toxin-containing endocytotic vesicles releases toxin into the cytoplasm, where it inhibits protein synthesis. The process of toxin transfer to the cytoplasm is presumed to account for the one-hour lag before toxin-induced inhibition of protein synthesis can be detected. Endocytotic uptake of toxin is impaired in resistant BW5147Ric^R.3 cells, and they are unaffected by toxin concentrations that inhibit protein synthesis and kill sensitive parental cells. Killing of resistant cells at low toxin concentrations was accomplished by encapsulating RCA_II into lipid vesicles capable of fusing with the plasma membrane. Direct introduction of toxin into resistant cells using lipid vesicles as carriers produced rapid inhibition (< 15 min) of protein synthesis and eliminated the lag in toxin action seen in sensitive cells exposed to free toxin. These findings are discussed in relation to the mechanism of toxin action and proposals that toxin activity requires structural modification of the toxin molecule at the cell surface before transport into the cell.     

Importance of glycolipid synthesis for butyric acid-induced sensitization to shiga toxin and intracellular sorting of toxin in A431 cells.

The human epidermoid carcinoma cell line A431 becomes highly sensitive to Shiga toxin upon treatment with butyric acid. This strong sensitization (> 1000-fold) is accompanied by an increase in the fraction of cell-associated toxin transported to the Golgi apparatus and to the endoplasmic reticulum (ER). Furthermore, our previous work showed that the length of the fatty acyl chain of Gb3, the Shiga toxin receptor, also was changed (longer fatty acids). We have not investigated the importance of this change by testing whether glycolipid synthesis is required for the changed intracellular sorting and the toxin sensitivity. We demonstrate here that inhibition of glycosphingolipid synthesis by inhibition of N-acyltransferase with fumonisin B1, by inhibition of glucosylceramide synthetase by PDMP or PPMP, or by inhibition of serine palmitoyl transferase by beta-fluoroalanine, inhibited the butyric acid-induced change in sensitivity and the increase in the fraction of cell-associated Shiga toxin transported to the Golgi apparatus and the ER. The block in butyric acid-induced sensitization caused by beta-fluoroalanine could be abolished by simultaneous addition of sphinganine or sphingosine. Thus, the data suggest that the fatty acyl chain length of glycosphingolipids is important for intracellular sorting and translocation of Shiga toxin to the cytosol.