Is cholesterol both the lock and key to abnormal transmembrane signals in Autism Spectrum Disorder?

Disturbances in cholesterol homeostasis have been associated with ASD. Lipid rafts are central in many transmembrane signaling pathways (including mTOR) and changes in raft cholesterol content affect their order function. Cholesterol levels are controlled by several mechanisms, including endoplasmic reticulum associated degradation (ERAD) of the rate limiting HMGCoA reductase. A new approach to increase cholesterol via temporary ERAD blockade using a benign bacterial toxin-derived competitor for the ERAD translocon is suggested.A new lock and key model for cholesterol/lipid raft dependent signaling is proposed in which the rafts provide both the afferent and efferent 'tumblers' across the membrane to allow 'lock and key' receptor transmembrane signals.

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.

Verotoxin Receptor-Based Pathology and Therapies.

Verotoxin, VT (aka Shiga toxin,Stx) is produced by enterohemorrhagic E. coli (EHEC) and is the key pathogenic factor in EHEC-induced hemolytic uremic syndrome (eHUS-hemolytic anemia/thrombocytopenia/glomerular infarct) which can follow gastrointestinal EHEC infection, particularly in children. This AB5 subunit toxin family bind target cell globotriaosyl ceramide (Gb3), a glycosphingolipid (GSL) (aka CD77, pk blood group antigen) of the globoseries of neutral GSLs, initiating lipid raft-dependent plasma membrane Gb3 clustering, membrane curvature, invagination, scission, endosomal trafficking, and retrograde traffic via the TGN to the Golgi, and ER. In the ER, A/B subunits separate and the A subunit hijacks the ER reverse translocon (dislocon-used to eliminate misfolded proteins-ER associated degradation-ERAD) for cytosolic access. This property has been used to devise toxoid-based therapy to temporarily block ERAD and rescue the mutant phenotype of several genetic protein misfolding diseases. The A subunit avoids cytosolic proteosomal degradation, to block protein synthesis via its RNA glycanase activity. In humans, Gb3 is primarily expressed in the kidney, particularly in the glomerular endothelial cells. Here, Gb3 is in lipid rafts (more ordered membrane domains which accumulate GSLs/cholesterol) whereas renal tubular Gb3 is in the non-raft membrane fraction, explaining the basic pathology of eHUS (glomerular endothelial infarct). Females are more susceptible and this correlates with higher renal Gb3 expression. HUS can be associated with encephalopathy, more commonly following verotoxin 2 exposure. Gb3 is expressed in the microvasculature of the brain. All members of the VT family bind Gb3, but with varying affinity. VT2e (pig edema toxin) binds Gb4 preferentially. Verotoxin-specific therapeutics based on chemical analogs of Gb3, though effective in vitro, have failed in vivo. While some analogs are effective in animal models, there are no good rodent models of eHUS since Gb3 is not expressed in rodent glomeruli. However, the mouse mimics the neurological symptoms more closely and provides an excellent tool to assess therapeutics. In addition to direct cytotoxicity, other factors including VT-induced cytokine release and aberrant complement cascade, are now appreciated as important in eHUS. Based on atypical HUS therapy, treatment of eHUS patients with anticomplement antibodies has proven effective in some cases. A recent switch using stem cells to try to reverse, rather than prevent VT induced pathology may prove a more effective methodology.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Glycosphingolipid storage in Fabry mice extends beyond globotriaosylceramide and is affected by ABCB1 depletion.

AIM: Fabry disease is caused by α-galactosidase A deficiency leading to accumulation of globotriaosylceramide (Gb3) in tissues. Clinical manifestations do not appear to correlate with total Gb3 levels. Studies examining tissue distribution of specific acyl chain species of Gb3 and upstream glycosphingolipids are lacking. MATERIAL & METHODS/RESULTS: Thorough characterization of the Fabry mouse sphingolipid profile by LC-MS revealed unique Gb3 acyl chain storage profiles. Storage extended beyond Gb3; all Fabry tissues also accumulated monohexosylceramides. Depletion of ABCB1 had a complex effect on glycosphingolipid storage. CONCLUSION: These data provide insights into how specific sphingolipid species correlate with one another and how these correlations change in the α-galactosidase A-deficient state, potentially leading to the identification of more specific biomarkers of Fabry disease.

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.

CD4(+) T-cells are unable to express the HIV natural resistance factor globotriosylceramide.

Globotriaosylceramide (Gb(3)) is a cell surface-expressed natural resistance factor for HIV infection, but, its expression in human T-cells remains unknown. Therefore, Gb(3) in resting or activated CD4(+) T-cells was assessed by flow cytometry and thin layer chromatography of cell extracts. We found the majority of CD4(+) T-cells, whether resting or activated, do not express Gb(3) at significant levels (<2% positive cells). Thus, HIV treatment or prevention strategies must focus on development of soluble Gb(3) analogues for inhibition of HIV infection.

Verotoxin A subunit protects lymphocytes and T cell lines against X4 HIV infection in vitro.

Our previous genetic, pharmacological and analogue protection studies identified the glycosphingolipid, Gb(3) (globotriaosylceramide, Pk blood group antigen) as a natural resistance factor for HIV infection. Gb(3) is a B cell marker (CD77), but a fraction of activated peripheral blood mononuclear cells (PBMCs) can also express Gb(3). Activated PBMCs predominantly comprise CD4+ T-cells, the primary HIV infection target. Gb(3) is the sole receptor for Escherichia coli verotoxins (VTs, Shiga toxins). VT1 contains a ribosome inactivating A subunit (VT1A) non-covalently associated with five smaller receptor-binding B subunits. The effect of VT on PHA/IL2-activated PBMC HIV susceptibility was determined. Following VT1 (or VT2) PBMC treatment during IL2/PHA activation, the small Gb(3)+/CD4+ T-cell subset was eliminated but, surprisingly, remaining CD4+ T-cell HIV-1(IIIB) (and HIV-1(Ba-L)) susceptibility was significantly reduced. The Gb(3)-Jurkat T-cell line was similarly protected by brief VT exposure prior to HIV-1(IIIB) infection. The efficacy of the VT1A subunit alone confirmed receptor independent protection. VT1 showed no binding or obvious Jurkat cell/PBMC effect. Protective VT1 concentrations reduced PBMC (but not Jurkat cell) proliferation by 50%. This may relate to the mechanism of action since HIV replication requires primary T-cell proliferation. Microarray analysis of VT1A-treated PBMCs indicated up regulation of 30 genes. Three of the top four were histone genes, suggesting HIV protection via reduced gene activation. VT blocked HDAC inhibitor enhancement of HIV infection, consistent with a histone-mediated mechanism. We speculate that VT1A may provide a benign approach to reduction of (X4 or R5) HIV cell susceptibility.

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.

Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane.

The influenza virus (IFV) acquires its envelope by budding from host cell plasma membranes. Using quantitative shotgun mass spectrometry, we determined the lipidomes of the host Madin-Darby canine kidney cell, its apical membrane, and the IFV budding from it. We found the apical membrane to be enriched in sphingolipids (SPs) and cholesterol, whereas glycerophospholipids were reduced, and storage lipids were depleted compared with the whole-cell membranes. The virus membrane exhibited a further enrichment of SPs and cholesterol compared with the donor membrane at the expense of phosphatidylcholines. Our data are consistent with and extend existing models of membrane raft-based biogenesis of the apical membrane and IFV envelope.

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).

Glycosphingolipid functions.

The combination of carbohydrate and lipid generates unusual molecules in which the two distinctive halves of the glycoconjugate influence the function of each other. Membrane glycolipids can act as primary receptors for carbohydrate binding proteins to mediate transmembrane signaling despite restriction to the outer bilayer leaflet. The extensive heterogeneity of the lipid moiety plays a significant, but still largely unknown, role in glycosphingolipid function. Potential interplay between glycolipids and their fatty acid isoforms, together with their preferential interaction with cholesterol, generates a complex mechanism for the regulation of their function in cellular physiology.

Adamantyl glycosphingolipids provide a new approach to the selective regulation of cellular glycosphingolipid metabolism.

Mammalian glycosphingolipid (GSL) precursor monohexosylceramides are either glucosyl- or galactosylceramide (GlcCer or GalCer). Most GSLs derive from GlcCer. Substitution of the GSL fatty acid with adamantane generates amphipathic mimics of increased water solubility, retaining receptor function. We have synthesized adamantyl GlcCer (adaGlcCer) and adamantyl GalCer (adaGalCer). AdaGlcCer and adaGalCer partition into cells to alter GSL metabolism. At low dose, adaGlcCer increased cellular GSLs by inhibition of glucocerebrosidase (GCC). Recombinant GCC was inhibited at pH 7 but not pH 5. In contrast, adaGalCer stimulated GCC at pH 5 but not pH 7 and, like adaGlcCer, corrected N370S mutant GCC traffic from the endoplasmic reticulum to lysosomes. AdaGalCer reduced GlcCer levels in normal and lysosomal storage disease (LSD) cells. At 40 µM adaGlcCer, lactosylceramide (LacCer) synthase inhibition depleted LacCer (and more complex GSLs), such that only GlcCer remained. In Vero cell microsomes, 40 µM adaGlcCer was converted to adaLacCer, and LacCer synthesis was inhibited. AdaGlcCer is the first cell LacCer synthase inhibitor. At 40 µM adaGalCer, cell synthesis of only Gb(3) and Gb(4) was significantly reduced, and a novel product, adamantyl digalactosylceramide (adaGb(2)), was generated, indicating substrate competition for Gb(3) synthase. AdaGalCer also inhibited cell sulfatide synthesis. Microsomal Gb(3) synthesis was inhibited by adaGalCer. Metabolic labeling of Gb(3) in Fabry LSD cells was selectively reduced by adaGalCer, and adaGb(2) was produced. AdaGb(2) in cells was 10-fold more effectively shed into the medium than the more polar Gb(3), providing an easily eliminated "safety valve" alternative to Gb(3) accumulation. Adamantyl monohexosyl ceramides thus provide new tools to selectively manipulate normal cellular GSL metabolism and reduce GSL accumulation in cells from LSD patients.

Cholesterol modulates glycolipid conformation and receptor activity.

We document a new dimension of surface recognition in which communication is controlled through the collective behavior of lipids. Membrane cholesterol induces a tilt in glycolipid receptor headgroup, resulting in loss of access for ligand binding. This property appears to organize erythrocyte blood group presentation and glycolipid receptor function during the activation of sperm fertility, suggesting that lipid 'allostery' is a means to regulate membrane recognition processes.