g(m1)-ganglioside and globotriaosylceramide

Patients with the autoimmune rheumatic disease systemic lupus erythematosus (SLE) have multiple defects in lymphocyte signaling and function that contribute to disease pathogenesis. Such defects could be attributed to alterations in metabolic processes, including abnormal control of lipid biosynthesis pathways. Here, we reveal that CD4+ T cells from SLE patients displayed an altered profile of lipid raft-associated glycosphingolipids (GSLs) compared with that of healthy controls. In particular, lactosylceramide, globotriaosylceramide (Gb3), and monosialotetrahexosylganglioside (GM1) levels were markedly increased. Elevated GSLs in SLE patients were associated with increased expression of liver X receptor β (LXRβ), a nuclear receptor that controls cellular lipid metabolism and trafficking and influences acquired immune responses. Stimulation of CD4+ T cells isolated from healthy donors with synthetic and endogenous LXR agonists promoted GSL expression, which was blocked by an LXR antagonist. Increased GSL expression in CD4+ T cells was associated with intracellular accumulation and accelerated trafficking of GSL, reminiscent of cells from patients with glycolipid storage diseases. Inhibition of GSL biosynthesis in vitro with a clinically approved inhibitor (N-butyldeoxynojirimycin) normalized GSL metabolism, corrected CD4+ T cell signaling and functional defects, and decreased anti-dsDNA antibody production by autologous B cells in SLE patients. Our data demonstrate that lipid metabolism defects contribute to SLE pathogenesis and suggest that targeting GSL biosynthesis restores T cell function in SLE.

Topics: 1-Deoxynojirimycin; Adult; Aged; Antigens, CD; B-Lymphocytes; CD4-Positive T-Lymphocytes; Female; Flow Cytometry; G(M1) Ganglioside; Gene Expression Regulation; Glycosphingolipids; Homeostasis; Humans; Lactosylceramides; Leukocytes, Mononuclear; Liver X Receptors; Lupus Erythematosus, Systemic; Lymphocyte Activation; Male; Membrane Microdomains; Middle Aged; Orphan Nuclear Receptors; Signal Transduction; Time Factors; Trihexosylceramides

Glycosphingolipids (GSL) on the surface of cells are important receptors in antigen/microbial recognition and cell adhesion. However, their functional characterization is often challenging. We have developed a catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for the identification of specific interactions between water-soluble proteins or protein complexes with GSL incorporated into nanodiscs. The specificity and sensitivity of the assay is demonstrated for interactions involving cholera toxin and Shiga toxin, with their natural GSL receptors, the ganglioside GM1, and the globotriaosylceramide Gb3, respectively. The detection of binding between cholera toxin and GM1 within a mixture of lipids extracted from cell membranes highlights the potential of this assay for the discovery of biologically relevant protein-GSL interactions.

Topics: Cell Membrane; Cholera Toxin; G(M1) Ganglioside; Glycosphingolipids; Protein Binding; Shiga Toxin; Spectrometry, Mass, Electrospray Ionization; Trihexosylceramides

Glycosphingolipids (GSLs) accumulate in cholesterol-enriched cell membrane domains and provide receptors for protein ligands. Lipid-based "aglycone" interactions can influence GSL carbohydrate epitope presentation. To evaluate this relationship, Verotoxin binding its receptor GSL, globotriaosyl ceramide (Gb(3)), was analyzed in simple GSL/cholesterol, detergent-resistant membrane vesicles by equilibrium density gradient centrifugation. Vesicles separated into two Gb(3/)cholesterol-containing populations. The lighter, minor fraction (<5% total GSL), bound VT1, VT2, IgG/IgM mAb anti-Gb(3), HIVgp120 or Bandeiraea simplicifolia lectin. Only IgM anti-Gb(3), more tolerant of carbohydrate modification, bound both vesicle fractions. Post-embedding cryo-immuno-EM confirmed these results. This appears to be a general GSL-cholesterol property, because similar receptor-inactive vesicles were separated for other GSL-protein ligand systems; cholera toxin (CTx)-GM1, HIVgp120-galactosyl ceramide/sulfatide. Inclusion of galactosyl or glucosyl ceramide (GalCer and GlcCer) rendered VT1-unreactive Gb(3)/cholesterol vesicles, VT1-reactive. We found GalCer and GlcCer bind Gb(3), suggesting GSL-GSL interaction can counter cholesterol masking of Gb(3). The similar separation of Vero cell membrane-derived vesicles into minor "binding," and major "non-binding" fractions when probed with VT1, CTx, or anti-SSEA4 (a human GSL stem cell marker), demonstrates potential physiological relevance. Cell membrane GSL masking was cholesterol- and actin-dependent. Cholesterol depletion of Vero and HeLa cells enabled differential VT1B subunit labeling of "available" and "cholesterol-masked" plasma membrane Gb(3) pools by fluorescence microscopy. Thus, the model GSL/cholesterol vesicle studies predicted two distinct membrane GSL formats, which were demonstrated within the plasma membrane of cultured cells. Cholesterol masking of most cell membrane GSLs may impinge many GSL receptor functions.

Topics: Animals; Cell Membrane; Chlorocebus aethiops; Cholera Toxin; Cholesterol; Cryoelectron Microscopy; G(M1) Ganglioside; Glycosphingolipids; HIV Envelope Protein gp120; Humans; Immunoblotting; Microscopy, Fluorescence; Microscopy, Immunoelectron; Protein Binding; Shiga Toxins; Trihexosylceramides; Vero Cells

Several bacterial and plant enterotoxin B subunit-islet autoantigen fusion proteins were compared for their ability to serve as islet autoantigen carriers and adjuvants for reduction of pancreatic islet inflammation associated with type 1 diabetes. The cholera toxin B subunit (CTB), the heat-labile toxin B subunit from enterotoxigenic Escherichia coli (LTB), the Shigella toxin B subunit (STB), and the plant toxin ricin B subunit (RTB) were genetically linked to the islet autoantigens proinsulin (INS) and glutamic acid decarboxylase (GAD). The adjuvant-autoantigen gene fusions were transferred to a bacterial expression vector and the corresponding fusion proteins synthesized in E. coli. The purified adjuvant-autoantigen proteins were fed to 5-wk-old nonobese diabetic (NOD) mice once a week for 4 wk. Histological examination of pancreatic islets isolated from inoculated mice showed significant levels of insulitis reduction in comparison with uninoculated mice. The ratio of serum anti-INS and anti-GAD IgG2c to IgG1 antibody isotype titers increased in all ligand-autoantigen inoculated animal groups, suggesting an increase in effector Th2 lymphocytes in B subunit-mediated insulitis suppression. The results of these experiments indicate that bacterial and plant enterotoxin B subunit ligand-autoantigens enhance insulitis reduction in NOD mice. This research prompts further exploration of a multiadjuvant/autoantigen co-delivery strategy that may facilitate type 1 diabetes prevention and suppression in animals and humans.

Topics: alpha-Fetoproteins; Animals; Antibody Formation; Asialoglycoproteins; Autoantigens; Bacterial Toxins; Cholera Toxin; Diabetes Mellitus, Type 1; Enterotoxins; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli Proteins; Female; Fetuins; G(M1) Ganglioside; Glutamate Decarboxylase; Immunoglobulin G; Immunoglobulin Isotypes; Immunotherapy, Active; Islets of Langerhans; Mice; Mice, Inbred NOD; Peptide Fragments; Proinsulin; Protein Subunits; Recombinant Fusion Proteins; Ricin; Shiga Toxin; Trihexosylceramides

Cholera and Shiga toxin bind to the cell surface via glycolipid receptors GM1 and Gb3, respectively. Surprisingly, the majority of Vero cells from a non-synchronized population bind either Cholera or Shiga toxin but not both toxins. The hypothesis that the differential expression of toxin receptors is regulated by the cell cycle was tested. We find that Cholera toxin binds preferentially in G0/G1, with little binding through S-phase to telophase, whereas Shiga toxin binds maximally through G2 to telophase but does not bind during G0/G1 and S-phase. The changes result from the corresponding changes in Gb3 and GM1 synthesis, not from variations of receptor transport to the cell surface. The changes do not reflect competition of Gb3 and GM1 synthesis for lactosylceramide. Cells as diverse as Vero cells, PC12 cells and astrocytes show the same cell-cycle-dependent regulation of glycosphingolipid receptors, suggesting that this novel phenomenon is based on a conserved regulatory mechanism.

Topics: Animals; Cell Cycle; Cells, Cultured; Chlorocebus aethiops; G(M1) Ganglioside; G1 Phase; G2 Phase; Hippocampus; Mice; Neurons; PC12 Cells; Rats; Receptors, Cell Surface; Resting Phase, Cell Cycle; Telophase; Trihexosylceramides; Vero Cells

Enterohemorrhagic Escherichia coli producing Shiga toxins 1 and/or 2 have become major foodborne pathogens. The specific binding of Shiga toxin 1 B-subunit to its receptor, a neutral glycolipid globotriaosylceramide Gb(3), on the apical surface of colonic epithelium followed by toxin entry into cells are the initial steps of the process, which can result in toxin transcytosis and systemic effects of infection including hemolytic uremic syndrome. Understanding the complex mechanisms of Shiga toxin 1 binding and internalization may help to develop new strategies directed at preventing toxin internalization. Fluorescence resonance energy transfer microscopy revealed the clustering of Shiga toxin receptors Gb(3) in lipid rafts with another glycosphingolipid G(M1) on the apical surface of highly polarized intestinal epithelial Caco-2 cells. Lipid rafts disruption significantly decreased internalization of Shiga toxin 1 B-subunit. Although disruption of lipid rafts by cholesterol depletion did not affect the amount of bound Shiga toxin 1 B-subunit, lipid rafts are necessary for toxin uptake across the apical membrane of Caco-2 cells.

Topics: Biological Transport; Caco-2 Cells; Cell Membrane; Cell Polarity; Cholesterol; Energy Transfer; Fluorescent Dyes; G(M1) Ganglioside; Humans; Membrane Microdomains; Microscopy, Fluorescence; Receptor Aggregation; Receptors, Cell Surface; Shiga Toxin 1; Trihexosylceramides

Topics: alpha-Galactosidase; beta-Galactosidase; Ceramides; Cerebroside-Sulfatase; Enzyme Activators; G(M1) Ganglioside; G(M2) Activator Protein; G(M2) Ganglioside; Galactosylgalactosylglucosylceramidase; Glucosylceramidase; Glucosylceramides; Glycoproteins; Glycosphingolipids; Hydrolases; Lysosomes; Protein Precursors; Proteins; Saposins; Sphingolipid Activator Proteins; Sulfoglycosphingolipids; Trihexosylceramides

Topics: Animals; Bacterial Toxins; Blood Platelets; Child; Chlorocebus aethiops; Cholera Toxin; Chromatography, Thin Layer; Endocytosis; Enterotoxins; Escherichia coli; Escherichia coli Proteins; Fluorescein-5-isothiocyanate; G(M1) Ganglioside; Glycosphingolipids; Humans; Kidney Glomerulus; Shiga Toxin; Shiga Toxins; Trihexosylceramides; Vero Cells

A small fraction of the molecules internalized by endocytosis reaches the Golgi complex through a retrograde pathway that is poorly understood. In the present work, we used bacterial toxins to study the retrograde pathway in Vero cells. The recombinant B subunit of verotoxin 1B (VT1B) was labeled with fluorescein to monitor its progress within the cell by confocal microscopy. This toxin, which binds specifically to the glycolipid globotriaosyl ceramide, entered endosomes by both clathrin-dependent and -independent pathways, reaching the Golgi complex. Once internalized, the toxin-receptor complex did not recycle back to the plasma membrane. The kinetics of internalization and the subcellular distribution of VT1B were virtually identical to those of another glycolipid-binding toxin, the B subunit of cholera toxin (CTB). Retrograde transport of VT1B and CTB was unaffected by addition of weak bases in combination with concanamycin, a vacuolar-type ATPase inhibitor. Ratio imaging confirmed that these agents neutralized the luminal pH of the compartments where the toxin was located. Therefore, the retrograde transport of glycolipids differs from that of proteins like furin and TGN38, which require an acidic luminal pH. Additional experiments indicated that the glycolipid receptors of VT1B and CTB are internalized independently and not as part of lipid "rafts" and that internalization is cytochalasin insensitive. We conclude that glycolipids utilize a unique, pH-independent retrograde pathway to reach compartments of the secretory system and that assembly of F-actin is not required for this process.

Topics: Animals; Bacterial Toxins; Biological Transport; Chlorocebus aethiops; Clathrin; Cytochalasin B; Cytotoxins; Enterotoxins; Escherichia coli; Furin; G(M1) Ganglioside; Glycolipids; Glycoproteins; Golgi Apparatus; Hydrogen-Ion Concentration; Membrane Glycoproteins; Membrane Proteins; Protein Conformation; Receptors, Cell Surface; Recombinant Proteins; Shiga Toxin 1; Subtilisins; Trihexosylceramides; Vero Cells; Vibrio cholerae

The activator protein for the enzymatic hydrolysis of sulfatide, ganglioside GM1, and globotriaosylceramide was purified from human kidney, brain, and urine. As far as they could be assayed, these three activities cochromatographed during all steps, indicating that they are due to the same protein. This result was corroborated by immunochemical comparison of individually purified activator preparations. In contrast, the activator for ganglioside GM2 hydrolysis could clearly be separated from the other activities. Kinetic data were determined for the interaction of the sulfatide activator with the different glycolipids and hydrolases.

Topics: Brain Chemistry; Chromatography, DEAE-Cellulose; Chromatography, High Pressure Liquid; Enzyme Activation; G(M1) Ganglioside; Globosides; Glycosphingolipids; Humans; Hydrolysis; Immunoelectrophoresis, Two-Dimensional; Isoelectric Focusing; Kidney; Sulfoglycosphingolipids; Trihexosylceramides

The conformations of the neutral glycosphingolipid, globotriaosylceramide, and of the methyl ester of GM1-ganglioside have been predicted by energy-minimization techniques and compared with those previously obtained for GM1- and GM2-ganglioside. A triple-binding-domain model is put forward to explain known specificities of binding between these glycosphingolipids and activator proteins. This model suggests that hydrophobic interactions, electrostatic interactions and hydrogen-bonding between sugar residues are important. The model is discussed in relation to previous studies on the effect of chemical modification of glycosphingolipids on their ligand properties.

Topics: G(M1) Ganglioside; Globosides; Glycoproteins; Glycosphingolipids; Models, Molecular; Protein Conformation; Proteins; Saposins; Sphingolipid Activator Proteins; Sulfoglycosphingolipids; Trihexosylceramides

Urine specimens from two sibs affected with cerebroside sulfatase activator deficiency were examined to ascertain whether the deficiency of the supplementary activator protein required for the enzymatic hydrolysis of cerebroside sulfate was also evident in urine. Material from chromatographic fractionations was examined for the activator activity to avoid ambiguities resulting from protein inhibition. There were substantial deficits in all chromatographic fractions corresponding to activator-containing fractions of control urines. Since patient urines contained elevated amounts of lactosylceramide, digalactosylceramide, and globotriaosylceramide and since similarities between activators for cerebroside sulfate and GM1 ganglioside hydrolyses had been noted previously, the chromatographic fractions were also examined for activators in other glycosphingolipid hydrolase systems. There was coincidence of activators for the GM1 ganglioside/beta-galactosidase and the globotriaosylceramide/alpha-galactosidase A reactions with the cerebroside sulfatase activator in control urine fractions, and the patients' urines were deficient in activator activities for the three reactions. Identity of the three activators was suggested and antiserum to purified GM1 ganglioside activator was used to test this possibility. There were depressed levels of cross-reacting material in fractions of patient urines by Ouchterlony double diffusion and in unfractionated urine by enzyme-linked immunosorbent assay. Purified activators for the cerebroside sulfate and GM1 ganglioside systems showed lines of identity with no spurring on Ouchterlony double diffusion, identical mobility on immunoelectrophoresis, and similar stimulatory activities toward hydrolysis of the three glycosphingolipid species by their respective enzymes. Finally, the three activator activities were retained by anti-GM1-activator IgG coupled to Sepharose 4B. The results suggest strongly that the same protein entity serves as activator for the enzymatic hydrolysis of cerebroside sulfate, GM1 ganglioside, and globotriaosylceramide.

Topics: alpha-Galactosidase; Animals; beta-Galactosidase; beta-N-Acetylhexosaminidases; Enzyme Activation; G(M1) Ganglioside; G(M2) Ganglioside; Gangliosides; Globosides; Glycosphingolipids; Hexosaminidases; Humans; Hydrolysis; Immunodiffusion; Immunosorbent Techniques; Protein Deficiency; Proteins; Proteinuria; Rats; Saposins; Trihexosylceramides

Functionally defined clones and lines of murine lymphocytes including myelomas, helper, suppressor and cytolytic T lymphocytes were analyzed for their glycosphingolipids (GSLs). GSLs were characterized by thin-layer chromatography and by high-performance liquid chromatography. Lymphocytes with different functions displayed, besides a number of common GSLs, some characteristic GSLs that may be regarded as markers. Globotriaosylceramide was found on myelomas and B blasts, whereas globotetraosylceramide was confined to helper T cells. All T cells including cytolytic T lymphocytes displayed gangliotetraosylceramide (asialo-GM1) as a major GSL, which was further characterized by sequential degradation with exoglycosidases.

Topics: Animals; Cell Line; Clone Cells; G(M1) Ganglioside; Globosides; Glycosphingolipids; Male; Mice; Mice, Inbred CBA; Mice, Inbred DBA; Multiple Myeloma; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory; Trihexosylceramides