ATP-binding cassette transporters mediate differential biosynthesis of glycosphingolipid species.

The cytosolic-oriented glucosylceramide (GlcCer) synthase is enigmatic, requiring nascent GlcCer translocation to the luminal Golgi membrane to access glycosphingolipid (GSL) anabolic glycosyltransferases. The mechanism by which GlcCer is flipped remains unclear. To investigate the role of GlcCer-binding partners in this process, we previously made cleavable, biotinylated, photoreactive GlcCer analogs in which the reactive nitrene was closely apposed to the GlcCer head group, while maintaining a C16-acyl chain. GlcCer-binding protein specificity was validated for both photoprobes. Using one probe, XLB, here we identified ATP-binding cassette (ABC) transporters ABCA3, ABCB4, and ABCB10 as unfractionated microsomal GlcCer-binding proteins in DU-145 prostate tumor cells. siRNA knockdown (KD) of these transporters differentially blocked GSL synthesis assessed in toto and via metabolic labeling. KD of ABCA3 reduced acid/neutral GSL levels, but increased those of LacCer, while KD of ABCB4 preferentially reduced neutral GSL levels, and KD of ABCB10 reduced levels of both neutral and acidic GSLs. Depletion of ABCA12, implicated in GlcCer transport, preferentially decreased neutral GSL levels, while ABCB1 KD preferentially reduced gangliosides, but increased neutral GSL Gb3. These results imply that multiple ABC transporters may provide distinct but overlapping GlcCer and LacCer pools within the Golgi lumen for anabolism of different GSL series by metabolic channeling. Differential ABC family member usage may fine-tune GSL biosynthesis depending on cell/tissue type. We conclude that ABC transporters provide a new tool for the regulation of GSL biosynthesis and serve as potential targets to reduce selected GSL species/subsets in diseases in which GSLs are dysregulated.

Therapeutic Uses of Bacterial Subunit Toxins.

The B subunit pentamer verotoxin (VT aka Shiga toxin-Stx) binding to its cellular glycosphingolipid (GSL) receptor, globotriaosyl ceramide (Gb3) mediates internalization and the subsequent receptor mediated retrograde intracellular traffic of the AB5 subunit holotoxin to the endoplasmic reticulum. Subunit separation and cytosolic A subunit transit via the ER retrotranslocon as a misfolded protein mimic, then inhibits protein synthesis to kill cells, which can cause hemolytic uremic syndrome clinically. This represents one of the most studied systems of prokaryotic hijacking of eukaryotic biology. Similarly, the interaction of cholera AB5 toxin with its GSL receptor, GM1 ganglioside, is the key component of the gastrointestinal pathogenesis of cholera and follows the same retrograde transport pathway for A subunit cytosol access. Although both VT and CT are the cause of major pathology worldwide, the toxin-receptor interaction is itself being manipulated to generate new approaches to control, rather than cause, disease. This arena comprises two areas: anti neoplasia, and protein misfolding diseases. CT/CTB subunit immunomodulatory function and anti-cancer toxin immunoconjugates will not be considered here. In the verotoxin case, it is clear that Gb3 (and VT targeting) is upregulated in many human cancers and that there is a relationship between GSL expression and cancer drug resistance. While both verotoxin and cholera toxin similarly hijack the intracellular ERAD quality control system of nascent protein folding, the more widespread cell expression of GM1 makes cholera the toxin of choice as the means to more widely utilise ERAD targeting to ameliorate genetic diseases of protein misfolding. Gb3 is primarily expressed in human renal tissue. Glomerular endothelial cells are the primary VT target but Gb3 is expressed in other endothelial beds, notably brain endothelial cells which can mediate the encephalopathy primarily associated with VT2-producing E. coli infection. The Gb3 levels can be regulated by cytokines released during EHEC infection, which complicate pathogenesis. Significantly Gb3 is upregulated in the neovasculature of many tumours, irrespective of tumour Gb3 status. Gb3 is markedly increased in pancreatic, ovarian, breast, testicular, renal, astrocytic, gastric, colorectal, cervical, sarcoma and meningeal cancer relative to the normal tissue. VT has been shown to be effective in mouse xenograft models of renal, astrocytoma, ovarian, colorectal, meningioma, and breast cancer. These studies are herein reviewed. Both CT and VT (and several other bacterial toxins) access the cell cytosol via cell surface ->ER transport. Once in the ER they interface with the protein folding homeostatic quality control pathway of the cell -ERAD, (ER associated degradation), which ensures that only correctly folded nascent proteins are allowed to progress to their cellular destinations. Misfolded proteins are translocated through the ER membrane and degraded by cytosolic proteosome. VT and CT A subunits have a C terminal misfolded protein mimic sequence to hijack this transporter to enter the cytosol. This interface between exogenous toxin and genetically encoded endogenous mutant misfolded proteins, provides a new therapeutic basis for the treatment of such genetic diseases, e.g., Cystic fibrosis, Gaucher disease, Krabbe disease, Fabry disease, Tay-Sachs disease and many more. Studies showing the efficacy of this approach in animal models of such diseases are presented.

Cardiac glycoside/aglycones inhibit HIV-1 gene expression by a mechanism requiring MEK1/2-ERK1/2 signaling.

The capacity of HIV-1 to develop resistance to current drugs calls for innovative strategies to control this infection. We aimed at developing novel inhibitors of HIV-1 replication by targeting viral RNA processing-a stage dependent on conserved host processes. We previously reported that digoxin is a potent inhibitor of this stage. Herein, we identify 12 other cardiac glycoside/aglycones or cardiotonic steroids (CSs) that impede HIV growth in HIV-infected T cells from clinical patients at IC50s (1.1-1.3 nM) that are 2-26 times below concentrations used in patients with heart conditions. We subsequently demonstrate that CSs inhibit HIV-1 gene expression in part through modulation of MEK1/2-ERK1/2 signaling via interaction with the Na+/K+-ATPase, independent of alterations in intracellular Ca2+. Supporting this hypothesis, depletion of the Na+/K+-ATPase or addition of a MEK1/2-ERK1/2 activator also impairs HIV-1 gene expression. Similar to digoxin, all CSs tested induce oversplicing of HIV-1 RNAs, reducing unspliced (Gag) and singly spliced RNAs (Env/p14-Tat) encoding essential HIV-1 structural/regulatory proteins. Furthermore, all CSs cause nuclear retention of genomic/unspliced RNAs, supporting viral RNA processing as the underlying mechanism for their disruption of HIV-1 replication. These findings call for further in vivo validation and supports the targeting of cellular processes to control HIV-1 infection.

Suppression of Adenovirus Replication by Cardiotonic Steroids.

The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies. IMPORTANCE: Despite human adenoviruses being a common and, in some instances, life-threating pathogen in humans, there are few well-tolerated therapies. In this report, we demonstrate that two cardiotonic steroids already in use in humans, digoxin and digitoxin, are potent inhibitors of multiple adenovirus species. A synthetic derivative of the cardiotonic steroid convallotoxin was even more potent than digoxin and digitoxin when tested with HAdV-C5. These drugs alter the cascade of adenovirus gene expression, acting after initiation of early gene expression to block viral DNA replication and synthesis of viral structural proteins. These findings validate a novel approach to treating adenovirus infections through the modulation of host cell processes.

Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases.

Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subunit has evolved to mimic a misfolded protein and hijack the ERAD membrane translocon (dislocon), to effect cytosolic access and cytopathology. We show such toxins compete for ERAD to rescue endogenous misfolded proteins. Cholera toxin or verotoxin (Shiga toxin) containing genetically inactivated (± an N-terminal polyleucine tail) A subunit can, within 2-4 hrs, temporarily increase F508delCFTR protein, the major cystic fibrosis (CF) mutant (5-10x), F508delCFTR Golgi maturation (<10x), cell surface expression (20x) and chloride transport (2x) in F508del CFTR transfected cells and patient-derived F508delCFTR bronchiolar epithelia, without apparent cytopathology. These toxoids also increase glucocerobrosidase (GCC) in N370SGCC Gaucher Disease fibroblasts (3x), another ERAD-exacerbated misfiling disease. We identify a new, potentially benign approach to the treatment of certain genetic protein misfolding diseases.

Synthesis of a novel photoactivatable glucosylceramide cross-linker.

The biosynthesis of glucosylceramide (GlcCer) is a key rate-limiting step in complex glycosphingolipid (GSL) biosynthesis. To further define interacting partners of GlcCer, we have made a cleavable, biotinylated, photoreactive GlcCer analog in which the reactive nitrene is closely apposed to the GlcCer head group, by substituting the native fatty acid with d, l-2-aminohexadecanoic acid. Two amino-GlcCer diastereomer cross-linkers (XLA and XLB) were generated. XLB proved an effective lactosylceramide (LacCer) synthase substrate while XLA was inhibitory. Both probes specifically bound and cross-linked the GlcCer binding protein, glycolipid transfer protein (GLTP), but not other GSL binding proteins (Shiga toxin and cholera toxin). GlcCer inhibited GLTP cross-linking. Both GlcCer cross-linkers competed with microsomal nitrobenzoxadiazole (NBD)-GlcCer anabolism to NBD-LacCer. GLTP showed marked, ATP-dependent enhancement of cell-free intact microsomal LacCer synthesis from endogenous or exogenous liposomal GlcCer, supporting a role in the transport/membrane translocation of cytosolic and extra-Golgi GlcCer. GLTP was specifically labeled by either XLA or XLB GlcCer cross-linker during this process, together with a (the same) small subset of microsomal proteins. These cross-linkers will serve to probe physiologically relevant GlcCer-interacting cellular proteins.

Inhibition of Rab prenylation by statins induces cellular glycosphingolipid remodeling.

Statins, which specifically inhibit HMG Co-A reductase, the rate-limiting step of cholesterol biosynthesis, are widely prescribed to reduce serum cholesterol and cardiac risk, but many other effects are seen. We now show an effect of these drugs to induce profound changes in the step-wise synthesis of glycosphingolipids (GSLs) in the Golgi. Glucosylceramide (GlcCer) was increased several-fold in all cell lines tested, demonstrating a widespread effect. Additionally, de novo or elevated lactotriaosylceramide (Lc3Cer; GlcNAcß1-3Galß1-4GlcCer) synthesis was observed in 70%. Western blot showed that GlcCer synthase (GCS) was elevated by statins, and GCS and Lc3Cer synthase (Lc3S) activities were increased; however, transcript was elevated for Lc3S only. Supplementation with the isoprenoid precursor, geranylgeranyl pyrophosphate (GGPP), a downstream product of HMG Co-A reductase, reversed statin-induced glycosyltransferase and GSL elevation. The Rab geranylgeranyl transferase inhibitor 3-PEHPC, but not specific inhibitors of farnesyl transferase, or geranylgeranyl transferase I, was sufficient to replicate statin-induced GlcCer and Lc3Cer synthesis, supporting a Rab prenylation-dependent mechanism. While total cholesterol was unaffected, the trans-Golgi network (TGN) cholesterol pool was dissipated and medial Golgi GCS partially relocated by statins. GSL-dependent vesicular retrograde transport of Verotoxin and cholera toxin to the Golgi/endoplasmic reticulum were blocked after statin or 3-PEHPC treatment, suggesting aberrant, prenylation-dependent vesicular traffic as a basis of glycosyltransferase increase and GSL remodeling. These in vitro studies indicate a previously unreported link between Rab prenylation and regulation of GCS activity and GlcCer metabolism.

Cholesterol masks membrane glycosphingolipid tumor-associated antigens to reduce their immunodetection in human cancer biopsies.

Glycosphingolipids (GSLs) are neoplastic and normal/cancer stem cell markers and GSL/cholesterol-containing membrane rafts are increased in cancer cell plasma membranes. We define a novel means by which cancer cells can restrict tumor-associated GSL immunoreactivity. The GSL-cholesterol complex reorients GSL carbohydrate to a membrane parallel, rather than perpendicular conformation, largely unavailable for antibody recognition. Methyl-ß-cyclodextrin cholesterol extraction of all primary human tumor frozen sections tested (ovarian, testicular, neuroblastoma, prostate, breast, colon, pheochromocytoma and ganglioneuroma), unmasked previously "invisible" membrane GSLs for immunodetection. In ovarian carcinoma, globotriaosyl ceramide (Gb3), the GSL receptor for the antineoplastic Escherichia coli-derived verotoxin, was increased throughout the tumor. In colon carcinoma, Gb3 detection was vastly increased within the neovasculature and perivascular stroma. In tumors considered Gb3 negative (neuroblastoma, Leydig testicular tumor and pheochromocytoma), neovascular Gb3 was unmasked. Tumor-associated GSL stage-specific embryonic antigen (SSEA)-1, SSEA-3, SSEA-4 and globoH were unmasked according to tumor: SSEA-1 in prostate/colon; SSEA-3 in prostate; SSEA-4 in pheochromocytoma/some colon tumors; globoH in prostate/some colon tumors. In colon, anti-SSEA-1 was tumor cell specific. Within the GSL-cholesterol complex, filipin-cholesterol binding was also reduced. These results may relate to the ill-defined benefit of statins on cancer prognosis, for example, prostate carcinoma. We found novel anti-tumor GSL antibodies circulating in 3/5 statin-treated, but not untreated, prostate cancer patients. Lowering tumor membrane cholesterol may permit immune recognition of otherwise unavailable tumor-associated GSL carbohydrate, for more effective immunosurveillance and active/passive immunotherapy. Our results show standard immunodetection of tumor GSLs significantly under assesses tumor membrane GSL content, impinging on the current use of such antigens as cancer vaccines.

Comparison of detection methods for cell surface globotriaosylceramide.

The cell surface-expressed glycosphingolipid (GSL), globotriaosylceramide (Gb(3)), is becoming increasingly important and is widely studied in the areas of verotoxin (VT)-mediated cytotoxicity, human immunodeficiency virus (HIV) infection, immunology and cancer. However, despite its diverse roles and implications, an optimized detection method for cell surface Gb(3) has not been determined. GSLs are differentially organized in the plasma membrane which can affect their availability for protein binding. To examine various detection methods for cell surface Gb(3), we compared four reagents for use in flow cytometry analysis. A natural ligand (VT1B) and three different monoclonal antibodies (mAbs) were optimized and tested on various human cell lines for Gb(3) detection. A differential detection pattern of cell surface Gb(3) expression, which was influenced by the choice of reagent, was observed. Two mAb were found to be suboptimal. However, two other methods were found to be useful as defined by their high percentage of positivity and mean fluorescence intensity (MFI) values. Rat IgM anti-Gb(3) mAb (clone 38-13) using phycoerythrin-conjugated secondary antibody was found to be the most specific detection method while the use of VT1B conjugated to Alexa488 fluorochrome was found to be the most sensitive; showing a rare crossreactivity only when Gb(4) expression was highly elevated. The findings of this study demonstrate the variability in detection of Gb(3) depending on the reagent and cell target used and emphasize the importance of selecting an optimal methodology in studies for the detection of cell surface expression of Gb(3).

Glycolipid-Dependent, Protease Sensitive Internalization of Pseudomonas aeruginosa Into Cultured Human Respiratory Epithelial Cells.

Internalization of PAK strain Pseudomonas aeruginosa into human respiratory epithelial cell lines and HeLa cervical cancer cells in vitro was readily demonstrable via a gentamycin protection assay. Depletion of target cell glycosphingolipids (GSLs) using a glucosyl ceramide synthase inhibitor, P4, completely prevented P. aeruginosa internalization. In contrast, P4 treatment had no effect on the internalization of Salmonella typhimurium into HeLa cells. Internalized P. aeruginosa were within membrane vacuoles, often containing microvesicles, between the bacterium and the limiting membrane. P. aeruginosa internalization was markedly enhanced by target cell pretreatment with the exogenous GSL, deacetyl gangliotetraosyl ceramide (Gg(4)). Gg(4) binds the lipid raft marker, GM1 ganglioside. Target cell pretreatment with TLCK, but not other (serine) protease inhibitors, prevented both P. aeruginosa host cell binding and internalization. NFkB inhibition also prevented internalization. A GSL-containing lipid-raft model of P. aeruginosa host cell binding/internalization is proposed.

A soluble sulfogalactosyl ceramide mimic promotes Delta F508 CFTR escape from endoplasmic reticulum associated degradation.

AdaSGC binds Hsc70s to inhibit ATPase activity. Using single-turnover assays, adaSGC, a soluble SGC mimic, preferentially inhibited Hsp40-activated Hsc70 ATP hydrolysis (Ki approximately 10 microM) to reduce C-terminal Hsc70-peptide binding and, potentially, chaperone function. ERAD of misfolded Delta F508 CFTR requires Hsc70-Hsp40 chaperones. In transfected baby hamster kidney (BHK) cells, adaSGC increased Delta F508CFTR ERAD escape, and after low-temperature glycerol rescue, maturation, and iodide efflux. Inhibition of SGC biosynthesis reduced Delta F508CFTR but not wtCFTR expression, whereas depletion of other glycosphingolipids had no affect. WtCFTR transfected BHK cells showed increased SGC synthesis compared with Delta F508CFTR/mock-transfected cells. Partial rescue of Delta F508CFTR by low-temperature glycerol increased SGC synthesis. AdaSGC also increased cellular endogenous SGC levels. SGC in the lung, liver, and kidney was severely depleted in Delta F508CFTR compared with wtCFTR mice, suggesting a role for CFTR in SGC biosynthesis.

Inhibition of Simian Virus 40 replication by targeting the molecular chaperone function and ATPase activity of T antigen.

Polyomaviruses such as BK virus and JC virus have been linked to several diseases, but treatments that thwart their propagation are limited in part because of slow growth and cumbersome culturing conditions. In contrast, the replication of one member of this family, Simian Virus 40 (SV40), is robust and has been well-characterized. SV40 replication requires two domains within the viral-encoded large tumor antigen (TAg): The ATPase domain and the N-terminal J domain, which stimulates the ATPase activity of the Hsp70 chaperone. To assess whether inhibitors of polyomavirus replication could be identified, we examined a recently described library of small molecules, some of which inhibit chaperone function. One compound, MAL2-11B, inhibited both TAg's endogenous ATPase activity and the TAg-mediated activation of Hsp70. MAL2-11B also reduced SV40 propagation in plaque assays and compromised DNA replication in cell culture and in vitro. Furthermore, the compound significantly reduced the growth of BK virus in a human kidney cell line. These data indicate that pharmacological inhibition of TAg's chaperone and ATPase activities may provide a route to combat polyomavirus-mediated disease.

Induction of HIV-1 resistance: cell susceptibility to infection is an inverse function of globotriaosyl ceramide levels.

To examine the role of the glycosphingolipid (GSL), globotriaosylceramide (Gb(3), CD77, p(k) blood group antigen) in HIV-1 infection, we have pharmacologically modulated Gb(3) metabolism in an X4 HIV-1 infectable monocytic cell line (THP-1) that naturally expresses Gb(3) and in a Gb(3)-expressing glioblastoma cell line (U87) transfected to express both CD4 and CCR5 to permit R5 HIV-1 infection. THP-1 and U87 cells were treated with either a competitive inhibitor of alpha-galactosidase A, 1-deoxygalactonojirimycin (DGJ) to induce Gb(3) accumulation, or a glucosylceramide synthase inhibitor, phenyl-2-palmitylamino-3-pyrrolidino-1-propanol (P4) to deplete cells of Gb(3). HIV susceptibility was determined via measurement of p24(gag) antigen production by ELISA. In addition, total cellular Gb(3) content was determined using thin layer chromatography followed by Verotoxin1 overlay binding. The cell surface expression of Gb(3) was verified by FACS analysis. We found that DGJ significantly decreased THP-1 and U87 cell susceptibility to HIV-1(IIIB) and HIV-1(BaL) infection, respectively, at a concentration of approximately 100 microM. In contrast, P4 (2 microM) substantially increased cellular susceptibility to HIV-1 infection. Total cellular GSL analysis verified increased Gb(3) expression in cells treated with DGJ and considerable reduction of Gb(3) in P4-treated cells as compared to controls. These results show a reciprocal relationship between Gb(3) expression and infection with either X4 HIV-1(IIIB) or R5 HIV-1(Ba-L). These results support previous studies that Gb(3) provides resistance to HIV infection. Variable Gb(3) expression may provide a natural HIV resistance factor in the general population, and pharmacological manipulation of Gb(3) levels may provide an approach to induction of HIV resistance.

Inhibition of multidrug resistance by adamantylgb3, a globotriaosylceramide analog.

Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.

Membrane cytosolic translocation of verotoxin A1 subunit in target cells.

In sensitive cells, verotoxin 1 (VT1) utilizes a globotriaosylceramide receptor-dependent retrograde transport pathway from the cell surface to the Golgi/endoplasmic reticulum (ER). The VT1 A subunit (VTA) is an RNA glycanase. Although translocation of VTA from the ER to the cytosol is considered the route for protein synthesis inhibition, cell-based evidence is lacking. A dual-fluorescent-labelled VT1 holotoxin was constructed to simultaneously monitor VTA and VT1 B subunit (VTB) intracellular transport. By confocal microscopy, VTA/VTB subunits remained associated throughout the retrograde transport pathway without cytosolic staining. However, in [125I]VT1-treated cells, the selective cytosolic translocation (4 %) of the activated form of VTA, VTA1, was demonstrated for the first time by monitoring [125I]VTA1 release after plasma membrane permeabilization by streptolysin O (SLO). Lactacystin, a proteasome inhibitor, increased cytosolic VTA1 and enhanced VT1 cytotoxicity. VT1 ER arrival coincided with cytosolic VTA1 detection. Brefeldin A and 16 degrees C, conditions which inhibit VT1 retrograde transport to the Golgi/ER, prevented VTA1 cytosolic translocation; however, these treatments did not completely prevent VT1-induced protein synthesis inhibition. Thus, efficient cytosolic translocation of VTA1 requires transport to the Golgi/ER, but alternative minor escape pathways for protein synthesis inhibition may operate when transport to the Golgi/ER is prevented. Inhibition of protein synthesis was time and dose dependent, and not necessarily a valid index of subsequent cytopathology. Only protein synthesis inhibition following >3 h VT1 exposure correlated with eventual cell cytotoxicity. Extrapolation of translocated cytosolic VTA1 values indicates that about one molecule of translocated VTA1 per cell is sufficient to inhibit protein synthesis and kill a cell.

Soluble adamantyl glycosphingolipid analogs as probes of glycosphingolipid function.

Despite the extensive structural characterization of glycosphingolipids (GSLs), their functions in cell physiology and pathobiology remain elusive. This is largely owing to the fact that they are difficult to handle, being insoluble in aqueous media, and that no one gene alone determines their synthesis. The heterogeneity of the lipid moiety provides a further confounding factor. GSLs are central components within lipid rafts, which are major foci for transmembrane signaling and interactions between eukaryotic cells and microbial pathogens. GSL receptor function often requires the lipid moiety, and lipid-free sugar analogs are ineffective inhibitors. In order to overcome some of these problems, we have synthesized adamantyl GSL analogs which, in part, mimic GSL membrane receptor function in solution. These compounds are made by replacing the endogenous fatty acid with an adamantan frame. This rigid hydrophobic structure surprisingly increases the water solubility of the conjugate and retains receptor function. These GSL mimics provide probes to study GSL receptor function within cells. They compete with native GSLs for ligand binding and are taken up by cells to potentially alter GSL-mediated interaction. We are focused on two derivatives, adamantyl globotriaosyl ceramide and adamantyl sulfogalactosyl ceramide, and have used these analogs to probe GSL function in microbial pathology and hsp70 function. This chapter describes the syntheses and uses of these mimics.

Laboratory and clinical Pseudomonas aeruginosa strains do not bind glycosphingolipids in vitro or during type IV pili-mediated initial host cell attachment.

The glycosphingolipids (GSLs) gangliotriaosylceramide (Gg(3)) and gangliotetraosylceramide (Gg(4)) have been implicated as receptors for type IV pili (T4P)-mediated Pseudomonas aeruginosa epithelial cell attachment. Since P. aeruginosa T4P are divided into five groups, the authors determined whether GSLs in general, and Gg(3) and Gg(4) in particular, are specifically bound and required for host epithelial cell attachment of clinical and laboratory strains within these groups. An enterohaemorrhagic Escherichia coli strain, CL56, known to bind to both Gg(3) and Gg(4), provided a positive control. TLC overlay showed no binding of more than 12 P. aeruginosa strains to either Gg(3) or Gg(4) (or other GSLs), while CL56 Gg(3)/Gg(4) binding was readily detectable. GSL ELISA similarly demonstrated no significant P. aeruginosa binding to Gg(3) or Gg(4), compared with CL56. Using a selective chemical inhibitor, epithelial cell GSL synthesis was abrogated, and Gg(3) and Gg(4) expression deleted, but P. aeruginosa attachment was not impaired. Target cell attachment was mediated by T4P, since non-piliated, but flagellated, mutants were unable to bind to the target cells. CFTR (cystic fibrosis transmembrane conductance regulator) has also been implicated as a receptor; however, in this work, overexpression of CFTR had no effect on P. aeruginosa binding. It is concluded that neither Gg(3) nor Gg(4) are specifically recognized by P. aeruginosa, and that endogenous GSLs do not have a role in the attachment of live intact P. aeruginosa to cultured lung epithelial cells. In contrast to whole piliated P. aeruginosa, T4P sheared from such bacteria showed significant Gg(3) and Gg(4) binding, which may explain the results of other studies.

Treatment of neutral glycosphingolipid lysosomal storage diseases via inhibition of the ABC drug transporter, MDR1. Cyclosporin A can lower serum and liver globotriaosyl ceramide levels in the Fabry mouse model.

We have shown that the ABC transporter, multiple drug resistance protein 1 (MDR1, P-glycoprotein) translocates glucosyl ceramide from the cytosolic to the luminal Golgi surface for neutral, but not acidic, glycosphingolipid (GSL) synthesis. Here we show that the MDR1 inhibitor, cyclosporin A (CsA) can deplete Gaucher lymphoid cell lines of accumulated glucosyl ceramide and Fabry cell lines of globotriaosyl ceramide (Gb3), by preventing de novo synthesis. In the Fabry mouse model, Gb3 is increased in the heart, liver, spleen, brain and kidney. The lack of renal glomerular Gb3 is retained, but the number of verotoxin 1 (VT1)-staining renal tubules, and VT1 tubular targeting in vivo, is markedly increased in Fabry mice. Adult Fabry mice were treated with alpha-galactosidase (enzyme-replacement therapy, ERT) to eliminate serum Gb3 and lower Gb3 levels in some tissues. Serum Gb3 was monitored using a VT1 ELISA during a post-ERT recovery phase +/- biweekly intra peritoneal CsA. After 9 weeks, tissue Gb3 content and localization were determined using VT1/TLC overlay and histochemistry. Serum Gb3 recovered to lower levels after CsA treatment. Gb3 was undetected in wild-type liver, and the levels of Gb3 (but not gangliosides) in Fabry mouse liver were significantly depleted by CsA treatment. VT1 liver histochemistry showed Gb3 accumulated in Kupffer cells, endothelial cell subsets within the central and portal vein and within the portal triad. Hepatic venule endothelial and Kupffer cell VT1 staining was considerably reduced by in vivo CsA treatment. We conclude that MDR1 inhibition warrants consideration as a novel adjunct treatment for neutral GSL storage diseases.

Lack of susceptibility of cells from patients with Fabry disease to productive infection with R5 human immunodeficiency virus.

A lack of viral replication after HIV-1Ba-L (R5) but not HIV-1IIIB (X4) infection was found using in-vitro activated peripheral blood-derived mononuclear cells from patients with Fabry disease, who have a defect in the catabolism of globotriaosylceramide. CCR5, but not CD4 or CXCR4 expression levels, were lower and the surface expression of globotriaosylceramide was negligible on activated patients' cells. Our findings suggest a novel resistance mechanism to productive infection with R5 HIV-1 that potentially involves abnormal globotriaosylceramide catabolism.

Role of multiple drug resistance protein 1 in neutral but not acidic glycosphingolipid biosynthesis.

Transfection studies have implicated the multiple drug resistance pump, MDR1, as a glucosyl ceramide translocase within the Golgi complex (Lala, P., Ito, S., and Lingwood, C. A. (2000) J. Biol. Chem. 275, 6246-6251). We now show that MDR1 inhibitors, cyclosporin A or ketoconazole, inhibit neutral glycosphingolipid biosynthesis in 11 of 12 cell lines tested. The exception, HeLa cells, do not express MDR1. Microsomal lactosyl ceramide and globotriaosyl ceramide synthesis from endogenous or exogenously added liposomal glucosyl ceramide was inhibited by cyclosporin A, consistent with a direct role for MDR1/glucosyl ceramide translocase activity in their synthesis. In contrast, cellular ganglioside synthesis in the same cells, was unaffected by MDR1 inhibition, suggesting neutral and acid glycosphingolipids are synthesized from distinct precursor glycosphingolipid pools. Metabolic labeling in wild type and knock-out (MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral glycosphingolipid (glucosyl ceramide, lactosyl ceramide) but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity. Cryo-immunoelectron microscopy showed MDR1 was predominantly intracellular, largely in rab6-containing Golgi vesicles and Golgi cisternae, the site of glycosphingolipid synthesis. These studies identify MDR1 as the major glucosyl ceramide flippase required for neutral glycosphingolipid anabolism and demonstrate a previously unappreciated dichotomy between neutral and acid glycosphingolipid synthesis.