Prominent immunogenicity of monosialosyl galactosylgloboside, carrying a stage-specific embryonic antigen-4 (SSEA-4) epitope in the ACHN human renal tubular cell line—a simple method for producing monoclonal antibodies against detergent-insoluble microdomains/raft

The binding of Shiga toxin (Stx) to Gb3Cer in detergent-insoluble microdomains (DIM)/raft of the ACHN human renal tubular cell line causes the temporal activation of the Src-family kinase Yes [1]. As a strategy for examining signaling mechanisms in DIM/raft, monoclonal antibodies (MAbs) are reliable tools for characterizing the constituent molecules in these microdomains. Thus, we employed DIM/raft suspensions of ACHN cells as an immunogen to develop MAbs. Simply subcutaneous injections of ACHN DIM/raft could elevate the serum titer after several boosts. The first screening was performed using dot-blot immunostaining with culture supernatants on a polyvinylidene difluoride (PVDF) membrane, on which DIM/raft or their chloroform/methanol (C/M) (2:1, v/v) extracts were dot-blotted. The next screening was performed by flowcytometric analysis of ACHN cells treated with or without a permeabilizing reagent. Many of the clones (21/31 clones=68%) thus obtained were also found to recognize to lipid fractions of the DIM/raft. Strikingly, all of the 21 clones that reacted to the lipid fraction were found to recognize monosialosyl galactosylgloboside (MSGG) or GL7, which carries the SSEA-4 epitope. Using DIM/raft as immunogens may enable us to easily obtain MAbs for glycolipids.     

ErbB-4 and TNF-alpha converting enzyme localization to membrane microdomains

Sequential proteolytic processing of ErbB-4 occurs in response to ligand addition. Here, we assess the localization of cleavable and non-cleavable ErbB-4 isoforms to membrane microdomains using three methodologies: (1) Triton X-100-insolubility, (2) Brij98-insolubility, and (3) detergent-free density gradient centrifugation. Whereas ErbB-4 translocated to a Triton X-100-insoluble fraction upon treatment of T47D cells with heregulin, it constitutively associated with a Brij98-insoluble fraction and a lipid raft fraction isolated using detergent-free methodology. Comparison of cleavable and non-cleavable isoforms of ErbB-4 revealed that both ErbB-4 isoforms are constitutively localized to either a Triton X-100-soluble or Brij98-insoluble fraction. In contrast, addition of heregulin resulted in translocation of the cleavable isoform to a detergent-free lipid raft. Tumor necrosis factor-alpha converting enzyme (TACE), the ectodomain secretase for ErbB-4, was present predominantly in its mature active form in most microdomains analyzed. These data suggest the assembly of ErbB-4 ectodomain cleavage apparatus in a membrane microdomain.     

Identification of low-density Triton X-100-insoluble plasma membrane microdomains in higher plants.

Low density Triton X-100-insoluble plasma membrane microdomains can be isolated from different mammalian cell types and are proposed to be involved in membrane trafficking, cell morphogenesis and signal transduction. Heterotrimeric G-proteins and their receptors are often associated with such domains, suggesting that these structures are involved in G-protein-coupled signaling. Here we report that detergent-insoluble plasma membrane microdomains also exist in higher plants and contain about 15% of membrane-bound heterotrimeric G-protein beta-subunit (Gbeta). Plasma membrane microdomains were isolated from tobacco leaves. They have low buoyant density relative to the surrounding plasma membrane, and are insoluble in Triton X-100 at 4 degrees C. Detergent-insoluble vesicles were examined by freeze-fracture electron microscopy. They have sizes in the range 100-400 nm, and often contain aggregated protein complexes. The majority of plasma membrane proteins cannot be detected in the Triton X-100-insoluble fraction, while few polypeptides are highly enriched. We identified six proteins with molecular masses of 22, 28, 35, 60, 67 and 94 kDa in detergent-insoluble fractions that are glycosylphosphatidylinositol (GPI)-anchored.     

Interaction of Shiga toxin from Escherichia coli with human intestinal epithelial cell lines and explants: Stx2 induces epithelial damage in organ culture

Shiga toxins (Stx) produced by Escherichia coli are associated with systemic complications such as haemolytic-uraemic syndrome. The mechanism of Stx translocation across the epithelial barrier is unknown as human intestinal epithelium lacks receptor Gb3. In this study, we have examined the interaction of purified Stx1 and 2 with Caco-2 (Gb3+) and T84 (Gb3-) cell lines, and determined the effects of Stx on human intestine using in vitro organ culture (IVOC). Stx exposure caused inhibition of protein synthesis and apoptosis in Caco-2 but not in T84 cells. However, both Stx1 and 2 were transported to the endoplasmic reticulum, and the Stx1 A-subunit was cleaved in a furin-dependent manner in both cell lines. Thus, a Gb3-independent retrograde transport route exists in T84 cells for Stx that does not induce cell damage. IVOC demonstrated increased epithelial cell extrusion in response to exposure to Stx2, but not Stx1, in both small intestine and colon. Pretreatment of Stx2 with Stx2-specific antibody abrogated this effect. Overlaying frozen sections with Stx showed lamina propria, but not epithelial, cell binding that paralleled Gb3 localization, and included endothelium and pericryptal myofibroblasts. This indicates that human intestinal epithelium may evince Stx2-induced damage in the absence of Gb3 receptors, by an as yet unrecognized mechanism.     

Evidence that cAMP-dependent protein kinase and a protein factor are involved in reactivation of triton X-100 models of sea urchin and starfish spermatozoa

A fraction obtained from detergent-extract of sea urchin or starfish spermatozoa using DEAE-cellulose chromatography reactivated Triton X-100 models of the spermatozoa in a cAMP-dependent manner. The DEAE fraction contained cAMP-dependent protein kinase with a high level of specific activity. Rabbit muscle inhibitor protein highly specific for cAMP-dependent protein kinases inhibited the ability of the deae fraction to induce reactivation of Triton X-100 models.l This inhibition paralleled inhibition of cAMP-dependent protein kinase activity of the DEAE fraction, suggesting participation of the enzyme in the cAMP-dependent reactivation of Triton X-100 models. However, cAMP-dependent protein kinase further purified from the DEAE fraction was incapable of reactivating these models by itself. A protein factor which was separated from the protein kinase in the course of purification of the enzyme was found to also be necessary for the reactivation. When cAMP-dependent protein kinase was pretreated with protein kinase inhibitor before addition of the protein factor, the reactivation of Triton X-100 models was no longer detected. However, after the protein factor had been incubated with cAMP and cAMP-dependent protein kinase, protein kinase inhibitor did not repress reactivation of Triton X-100 models. We propose that the reactivation needs phosphorylation of the protein factor by cAMP-dependent protein kinase.     

Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains

Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell type. Methyl-β-cyclodextrin (MβCD)-mediated cholesterol depletion resulted in only partial destruction of lipid rafts, accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types.     

Escherichia coli Shiga toxins induce apoptosis in epithelial cells that is regulated by the Bcl-2 family

Human intestinal cells lack globotriaosylceramide (Gb(3)), the receptor for Shiga toxin-1 (Stx1) and Shiga toxin-2 (Stx2). Therefore, the role of these toxins in mediating intestinal disease during infection with Shiga toxin-producing Escherichia coli is unclear. The aims of this study were to determine whether Stx1 and Stx2 induce apoptosis in epithelial cells expressing (HEp-2, Caco-2) or lacking (T84) Gb(3) and to characterize the role of the Bcl-2 family. Stx1 (12.5 ng/ml) induced apoptosis in both HEp-2 (21.9 +/- 7.9% vs. 0.8 +/- 0.3%, P = 0.01) and Caco-2 (10.1 +/- 1.2% vs. 3.1 +/- 0.4%, P = 0.006) cells but not in Gb(3)-deficient T84 cells. Toxin-mediated apoptosis of HEp-2 cells was associated with enhanced expression of the proapoptotic protein Bax. Inhibition of caspase activation prevented toxin-stimulated apoptosis. In addition, overexpression of Bcl-2 by transient transfection blocked Stx1-stimulated cell death. These findings indicate that Shiga toxins produced by E. coli signal Gb(3)-expressing epithelial cells to undergo apoptosis in association with enhanced Bax expression, thereby resulting in activation of the caspase cascade.     

Uncommon membrane distribution of Shiga toxin glycosphingolipid receptors in toxin-sensitive human glomerular microvascular endothelial cells

Membrane microdomain association of the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), the highly and less effective receptors, respectively, for Shiga toxins (Stxs), is assumed as a functional requirement for Stx-mediated cytotoxicity. In a previous study, we demonstrated predominant localization of Stx receptors in cholesterol-enriched membrane microdomains of moderately Stx-sensitive human brain microvascular endothelial cells (HBMECs) by means of detergent-resistant membranes (DRMs). Here we report a different preferential distribution of Stx receptors in non-DRM fractions of human glomerular microvascular endothelial cells (GMVECs), the major targets of Stxs in the human kidney. Full structural characterization of Stx receptors using electrospray ionization (ESI) mass spectrometry revealed Gb3Cer and Gb4Cer lipoforms with ceramide moieties mainly composed of C24:0/C24:1 or C16:0 fatty acid and sphingosine (d18:1) in GMVECs comparable to those previously found in HBMECs. Thin-layer chromatography immunostaining demonstrated an approximately 2-fold higher content of Gb3Cer and a 1.4-fold higher content of Gb4Cer in GMVECs than in HBMECs. However, this does not explain the remarkable higher cytotoxic action of Stx1 and Stx2 toward GMVECs as compared with HBMECs. Our finding opens new questions on the microdomain association of Stx receptors and the functional role of GSLs in the membrane assembly of GMVECs.     

Role of the FYVE finger and the RUN domain for the subcellular localization of Rabip4

Rabip4 is a Rab4 effector, which possesses a RUN domain, two coiled-coil domains, and a FYVE finger. It is associated with the early endosomes and leads, in concert with Rab4, to the enlargement of endosomes, resulting in the fusion of sorting and recycling endosomes. Our goal was to characterize the role of these various domains in Rabip4 subcellular localization and their function in Chinese hamster ovary cells. Although the FYVE finger domain specifically bound phosphatidylinositol 3-phosphate and was necessary for the function of Rabip4, it was not sufficient for the protein association with membranes. Indeed a protein containing the FYVE finger and the Rab4-binding site was cytosolic, whereas the total protein was mostly associated to the membrane fraction, whether or not cells were pretreated with wortmannin. By contrast, a construct corresponding to the N-terminal end, Rabip4-(1-212), and containing the RUN domain was membrane-associated. The complete protein partitioned between the Triton X-100-insoluble and -soluble fractions and a wortmannin treatment increased the amount of the protein in the Triton X-100 fraction. Rabip4-(1-212) was totally Triton X-100-insoluble, and confocal microscopic examination showed that it labeled not only the endosomes, positive for Rabip4, but also a filamentous network with a honeycomb appearance. The Triton X-100-insoluble fraction that contains Rabip4 did not correspond to the caveolin or glycosylphosphatidylinositol-enriched lipid rafts. Rabip4 did not appear directly linked to actin but seemed associated to the actin network. We propose that the subcellular localization of the protein is primarily driven by the RUN domain to endosomal microdomains characterized by Triton X-100 insolubility and that the FYVE domain and the Rab4-binding domain then allow for the recruitment of the protein to lipophilic microdomains enriched in phosphatidylinositol 3-phosphate.     

Shiga toxin binding to glycolipids and glycans

Background

Immunologically distinct forms of Shiga toxin (Stx1 and Stx2) display different potencies and disease outcomes, likely due to differences in host cell binding. The glycolipid globotriaosylceramide (Gb3) has been reported to be the receptor for both toxins. While there is considerable data to suggest that Gb3 can bind Stx1, binding of Stx2 to Gb3 is variable.

Methodology

We used isothermal titration calorimetry (ITC) and enzyme-linked immunosorbent assay (ELISA) to examine binding of Stx1 and Stx2 to various glycans, glycosphingolipids, and glycosphingolipid mixtures in the presence or absence of membrane components, phosphatidylcholine, and cholesterol. We have also assessed the ability of glycolipids mixtures to neutralize Stx-mediated inhibition of protein synthesis in Vero kidney cells.

Results

By ITC, Stx1 bound both Pk (the trisaccharide on Gb3) and P (the tetrasaccharide on globotetraosylceramide, Gb4), while Stx2 did not bind to either glycan. Binding to neutral glycolipids individually and in combination was assessed by ELISA. Stx1 bound to glycolipids Gb3 and Gb4, and Gb3 mixed with other neural glycolipids, while Stx2 only bound to Gb3 mixtures. In the presence of phosphatidylcholine and cholesterol, both Stx1 and Stx2 bound well to Gb3 or Gb4 alone or mixed with other neutral glycolipids. Pre-incubation with Gb3 in the presence of phosphatidylcholine and cholesterol neutralized Stx1, but not Stx2 toxicity to Vero cells.

Conclusions

Stx1 binds primarily to the glycan, but Stx2 binding is influenced by residues in the ceramide portion of Gb3 and the lipid environment. Nanomolar affinities were obtained for both toxins to immobilized glycolipids mixtures, while the effective dose for 50% inhibition (ED₅₀) of protein synthesis was about 10⁻¹¹ M. The failure of preincubation with Gb3 to protect cells from Stx2 suggests that in addition to glycolipid expression, other cellular components contribute to toxin potency.     

P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains

P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) superfamily responsible for the ATP-driven extrusion of diverse hydrophobic molecules from cells, is a cause of multidrug resistance in human tumours. Pgp can also operate as a phospholipid and glycosphingolipid flippase, and has been functionally linked to cholesterol, suggesting that it might be associated with sphingolipid-cholesterol microdomains in cell membranes. We have used nonionic detergent extraction and density gradient centrifugation of extracts from the multidrug-resistant Chinese hamster ovary cell line, CH(R)B30, to address this question. Our data indicate that Pgp is localized in intermediate-density membrane microdomains different from classical lipid rafts enriched in Src-family kinases. We demonstrate that Brij-96 can selectively isolate the Pgp domains, separating them from the caveolar and classical lipid rafts. Pgp was found entirely in the Brij-96-insoluble domains, and only partially in the Triton X-100-insoluble membrane microdomains. We studied the sensitivity of these domains to cholesterol removal, as well as their relationship to GM(1) ganglioside- and caveolin-1-enriched caveolar domains. We found that the buoyant density of the Brij-96-based Pgp-containing microdomains was sensitive to cholesterol removal by methyl-beta-cyclodextrin. The Brij-96 domains retained their structural integrity after cholesterol depletion while, in contrast, the Triton X-100-based caveolin-1/GM(1) microdomains did not. Using confocal fluorescence microscopy, we determined that caveolin-1 and GM(1) colocalized, while Pgp and caveolin-1, or Pgp and GM(1), did not. Our results suggest that Pgp does not interact directly with caveolin-1, and is localized in intermediate-density domains, distinct from classical lipid rafts and caveolae, which can be isolated using Brij-96.     

Characterization of clathrin and Syk interaction upon Shiga toxin binding

Shiga toxin (Stx) is a bacterial toxin that binds to its receptor Gb3 at the plasma membrane. It is taken up by endocytosis and transported retrogradely via the Golgi apparatus to the endoplasmic reticulum. The toxin is then translocated to the cytosol where it exerts its toxic effect. We have previously shown that phosphorylation of clathrin heavy chain (CHC) is an early event following Stx binding to HeLa cells, and that this requires the activity of the tyrosine kinase Syk. Here, we have investigated this event in more detail in the B lymphoid cell line Ramos, which expresses high endogenous levels of both Syk and Gb3. We report that efficient endocytosis of Stx in Ramos cells requires Syk activity and that Syk is recruited to the uptake site of Stx. Furthermore, in response to Stx treatment, CHC and Syk were rapidly phosphorylated in a Src family kinase dependent manner at Y1477 and Y352, respectively. We show that these phosphorylated residues act as binding sites for the direct interaction between Syk and CHC. Interestingly, Syk–CHC complex formation could be induced by both Stx and B cell receptor stimulation.     

Kinetic analysis of binding between Shiga toxin and receptor glycolipid Gb3Cer by surface plasmon resonance

Shiga toxin (Stx) binds to the receptor glycolipid Gb3Cer on the cell surface and is responsible for hemolytic uremic syndrome. Stx has two isoforms, Stx1 and Stx2, and in clinical settings Stx2 is known to cause more severe symptoms, although the differences between the mechanisms of action of Stx1 and Stx2 are as yet unknown. In this study, the binding modes of these two isoforms to the receptor were investigated with a surface plasmon resonance analyzer to compare differences by real time receptor binding analysis. A sensor chip having a lipophilically modified dextran matrix or quasicrystalline hydrophobic layer was used to immobilize an amphipathic lipid layer that mimics the plasma membrane surface. Dose responsiveness was observed with both isoforms when either the toxin concentration or the Gb3Cer concentration was increased. In addition, this assay was shown to be specific, because neither Stx1 nor Stx2 bound to GM3, but both bound weakly to Gb4Cer. It was also shown that a number of fitting models can be used to analyze the sensorgrams obtained with different concentrations of the toxins, and the "bivalent analyte" model was found to best fit the interaction between Stxs and Gb3Cer. This shows that the interaction between Stxs and Gb3Cer in the lipid bilayer has a multivalent effect. The presence of cholesterol in the lipid bilayer significantly enhanced the binding of Stxs to Gb3Cer, although kinetics were unaffected. The association and dissociation rate constants of Stx1 were larger than those of Stx2: Stx2 binds to the receptor more slowly than Stx1 but, once bound, is difficult to dissociate. The data described herein clearly demonstrate differences between the binding properties of Stx1 and Stx2 and may facilitate understanding of the differences in clinical manifestations caused by these toxins.     

Effects of gangliosides GM3 and De-N-acetyl GM3 on epidermal growth factor receptor kinase activity and cell growth.

Previously it was reported (Bremer, E.G., Schlessinger, J., and Hakomori, S.-I. (1986) J. Biol. Chem. 261, 2434-2440) that ganglioside GM3 inhibited epidermal growth factor (EGF)-stimulated phosphorylation of the EGF receptor in Triton X-100-treated preparations of human epidermoid carcinoma (A431) cell membranes. In addition, these authors reported that GM3 inhibited the growth of A431 cells. In contrast, a modified ganglioside, de-N-acetyl GM3, enhanced the EGF-dependent tyrosine kinase activity of the EGF receptor. In this work and in subsequent studies (Hanai, N., Dohi, T., Nores, G. A., and Hakomori, S.-I. (1988) J. Biol. Chem. 263, 6296-6301), the tyrosine kinase activity of the receptor from A431 cell membranes was assayed in the presence of Triton X-100. In this report, we confirm that GM3 inhibited and de-N-acetyl GM3 stimulated EGF receptor autophosphorylation in the presence of Triton X-100. However, in the absence of detergents, ganglioside GM3 inhibited EGF-stimulated receptor autophosphorylation, whereas de-N-acetyl GM3 had no effect on EGF-stimulated receptor autophosphorylation. The effects of these gangliosides on receptor autophosphorylation were measured in both A431 cell plasma membranes and in 3T3 cell membranes permeabilized to [32P]ATP by a freeze-thaw procedure, in intact A431 cells permeabilized with alamethicin, and in intact A431 cells grown in the presence of [32P]orthophosphate. Thus, the inhibitory effect of GM3 on receptor autophosphorylation was demonstrated in the presence and in the absence of detergent; the stimulatory effect of de-N-acetyl GM3 was observed only in the presence of detergent. We also demonstrate that ganglioside GM3 inhibited EGF-stimulated growth of transfected murine fibroblasts (3T3) that express the gene for human EGF receptor (Velu, T. J., Beguinot, L., Vass, W. C., Zhang, K., Pastan, I., and Lowy, D. R. (1989) J. Cell. Biochem. 39, 153-166). De-N-acetyl ganglioside GM3 had no effect on the growth of these cells. Growth of control fibroblasts, which lack endogenous EGF receptors (Pruss, R. M., and Herschman, H. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3918-3921), was not affected by the presence of either ganglioside. Similarly, ganglioside GM3, but not de-N-acetyl ganglioside GM3, inhibited the EGF-dependent incorporation of [3H]thymidine into DNA by transfected fibroblasts. Incorporation of labeled thymidine into DNA of control fibroblasts was not affected by the presence of either ganglioside. These studies indicate that ganglioside GM3, but not its deacetylated analogue, can affect EGF receptor kinase activity in intact membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

The novel neutrophil differentiation marker phosphatidylglucoside mediates neutrophil apoptosis

A new type of glycolipid, phosphatidylglucoside (PtdGlc), was identified as a component of raft-like membrane domains of the human leukemia cell line HL-60. In this study, we show that PtdGlc forms functional domains that are different from those produced by lactosylceramide (LacCer)-enriched lipid rafts. These rafts initiate neutrophil apoptosis. Neutrophils are the only type of human peripheral blood leukocyte or monocyte-derived dendritic cell to express large amounts of PtdGlc on their cell surfaces. PtdGlc was not colocalized with LacCer. Anti-PtdGlc IgM DIM21 did not induce neutrophil chemotaxis or superoxide generation, whereas anti-LacCer IgM T5A7 induced these activities. DIM21, but not T5A7, significantly induced neutrophil apoptosis. DIM21-induced apoptosis was inhibited by specific inhibitors of cysteine-containing aspartate-specific proteases (caspases)-8, -9, and -3 but not by the Src family kinase inhibitor PP1, PIP(3) kinase inhibitor LY294002, NADPH oxidase inhibitor diphenyleneiodonium, superoxide dismutase, or catalase. PtdGlc was colocalized with Fas on the neutrophil plasma membrane. DIM21 and the agonist anti-Fas Ab DX2 induced the formation of large Fas-colocalized clusters of PtdGlc on the plasma membrane. Furthermore, the antagonistic anti-Fas Ab ZB4 significantly inhibited DIM21-induced neutrophil apoptosis. These results suggest that PtdGlc is specifically expressed on neutrophils and mediates apoptosis of these cells, and that the Fas-associated death signal may be involved in PtdGlc-mediated apoptosis.     

An association of 27- and 40-kDa molecules with glycolipids that bind A-B bacterial enterotoxins to cultured cells

It is well recognized that the Shiga-like toxins (Stxs) preferentially bind to Gb3 glycolipids and the cholera toxin (CT) and heat-labile enterotoxin (LTp) bind to GM1 gangliosides. After binding to the cell surface, A-B bacterial enterotoxins have to be internalized by endocytosis. The transport of the toxin-glycolipid complex has been documented in several manners but the actual mechanisms are yet to be clarified. We applied a heterobifunctional cross-linker, sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropionate (SASD), to detect the membrane proteins involved in the binding and the transport of A-B bacterial enterotoxins in cultured cells. Both Stx1 and Stx2 bound to the detergent-insoluble microdomain (DIM) of Vero cells and Caco-2 cells, which were susceptible to the toxin, but neither was bound to insusceptible CHO-K1 cells. Both CT and LTp bound to the DIM of Vero cells, Caco-2 cells, and CHO-K1 cells. In a cross-linking experiment, Stx1 cross-linked only with a 27-kDa molecule, while Stx2, which was more potently toxic than Stx1, cross-linked with 27- and 40-kDa molecules of Vero cells as well as of Caco-2 cells; moreover, no molecules were cross-linked with the insusceptible CHO-K1 cells. LTp was cross-linked only to the 27-kDa molecule of these three cell types but the CT, which was more toxic than LTp, was also cross-linked with 27- and 40-kDa molecules of Vero cells, Caco-2 cells, and CHO-K1 cells. The 27- and the 40-kDa molecules might play a role in the endocytosis and retrograde transport of A-B bacterial enterotoxins.     

Latrunculin B facilitates Shiga toxin 1 transcellular transcytosis across T84 intestinal epithelial cells

Shiga toxins (Stx), released into the intestinal lumen by enterohemorrhagic Escherichia coli (EHEC), are major virulence factors responsible for gastrointestinal and systemic illnesses. These pathologies are believed to be due to the action of the toxins on endothelial cells, which express the Stx receptor, the glycosphingolipid Gb3. To reach the endothelial cells, Stx must translocate across the intestinal epithelial monolayer. This process is poorly understood. We investigated Stx1 movement across the intestinal epithelial T84 cell model and the role of actin turnover in this transcytosis. We showed that changes in the actin cytoskeleton due to latrunculin B, but not cytochalasin D or jasplakinolide, significantly facilitate toxin transcytosis across T84 monolayers. This trafficking is transcellular and completely inhibited by tannic acid, a cell impermeable plasma membrane fixative. This indicates that actin turnover could play an important role in Stx1 transcellular transcytosis across intestinal epithelium in vitro. Since EHEC attachment to epithelial cells causes an actin rearrangement, this finding may be highly relevant to Stx-induced disease.     

Receptor kinases with leucine-rich repeats are enriched in Triton X-100 insoluble plasma membrane microdomains from plants

In mammals, signalling components at the cell surface are clustered in Triton X-100 insoluble plasma membrane microdomains. We isolated plasma membrane microdomains from Arabidopsis and mustard cotyledons and determined their protein composition by mass spectrometry. Although the protein composition of the plant vesicles differ from the composition of the animal vesicles, they are also enriched in signalling components. We identified at least seven receptor kinases with leucine-rich repeats, 10 other kinases, the β subunit of heterotrimeric G-proteins and five small GTP-binding proteins. Thus, specific signalling components are highly enriched in plant plasma membrane microdomains while others are excluded.     

Characterization of a shiga-toxin 1-resistant stock of vero cells

Shiga toxins (Stxs, also referred to as verotoxins) were first described as a novel cytotoxic activity against Vero cells. In this study, we report the characterization of an Stx1-resistant (R-) stock of Vero cells. (1) When the susceptibility of R-Vero cells to Stx1 cytotoxicity was compared to that of Stx1-sensitive (S-) Vero cells by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, cell viability after 48-hr exposure to 10 pg/ml of Stx1 was greater than 80% and less than 15%, respectively. (2) Although both a binding assay of fluorescence-labeled Stx1 and lipid analysis indicated considerable expression of Gb3Cer, a functional receptor for Stxs, in both Vero cells, anti-Gb3Cer monoclonal antibodies capable of binding to S-Vero cells failed to effectively label R-Vero cells, suggesting a conformational difference in the Gb3Cer expressed on R-Vero cells. (3) The lipid analysis also showed that the R-Vero cells contained significant amounts of Gb4Cer. In addition, introduction of exogenous Gb4Cer into S-Vero cells slightly inhibited Stx1 cytotoxicity, suggesting some correlation between glycosphingolipid composition and Stx1 resistance. (4) Both butyrate treatment and serum depression eliminated the Stx1 resistance of R-Vero cells. (5) The results of the analysis by confocal microscopy suggest a difference in intracellular transport of Stx1 between R-Vero and S-Vero cells. Further study of R-Vero cells may provide a model of Stx1 resistance via distinct intracellular transport of Stx1.     

Ca2+/calmodulin-dependent protein phosphorylation associated with the cytoskeleton of quiescent rat fibroblast (3Y1) cells.

Endogenous phosphorylation of the crude membrane fraction of cultured 3Y1 fibroblast cells was enhanced by the addition of Ca2+/calmodulin. Both Ca2+/calmodulin-dependent protein kinase activity and its substrate were present in a cytoskeletal fraction, obtained as a pellet after washing of the membrane fraction with 2 mM EGTA, 0.6 M NaCl, and 1% Triton X-100. The phosphorylatable protein in the Triton X-insoluble fraction was identified by immunoblotting as vimentin. This endogenous phosphorylation induced by calmodulin was inhibited by the addition of KN-62, a specific Ca2+/calmodulin-dependent protein kinase II inhibitor, in a dose-dependent manner. However, phosphorylation of the 59 kDa protein (vimentin) in this fraction was not stimulated by adding both phosphatidyl serine and cAMP, thereby suggesting the absence of protein kinase C or of cAMP-dependent protein kinase in this fraction. The protein kinase associated with the Triton X-insoluble fraction phosphorylated the Ca2+/calmodulin-dependent protein kinase II-specific site of synapsin I from the bovine cortex. Two-dimensional phosphopeptide maps of vimentin indicated that a major phosphopeptide phosphorylated by the endogenous calmodulin-dependent kinase also appears to be the same as a major phosphopeptide phosphorylated by the exogenous Ca2+/calmodulin-dependent protein kinase II. Our results suggest that cytoskeleton-associated Ca2+/calmodulin-dependent protein kinase II regulates dynamic cellular functions through the phosphorylation of cytoskeletal elements in non-neural cells.