Globotriaosylceramide Research Articles

Universal Method for the Facile Production of Glycolipid/Lipid Matrices for the Affinity Purification of Binding Ligands

Glycolipid recognition is a common motif in cellular physiology and bacterial pathogenesis. Such protein/lipid interactions are most conveniently demonstrated by the thin-layer chromatogram overlay. We have designed a simple affinity matrix for the purification of such glycolipid (or lipid) binding ligands based on the same principle, i.e., glycolipid (or lipid) adsorbed onto silica. The versatility of the procedure is demonstrated by the purification of several anti-glycolipid antibodies and anti-phosphatidyl ethanolamine (anti-PE) and the affinity purification of the Escherichia coli-derived verotoxin which binds to globotriaosyl ceramide.

Verotoxin-binding in human renal sections.

Gastrointestinal infection with verotoxin-producing Escherichia coli (VTEC) has been strongly implicated in the etiology of the hemolytic uremic syndrome (HUS), the leading cause of pediatric acute renal failure. The binding of fluorescein-conjugated VT1 overlaid on to frozen human renal sections has been examined. Sections from biopsies of infants aged < 2 years were compared with those from adult autopsies. VT primarily stained distal convoluted tubules, particularly those adjacent to glomeruli, and collecting ducts. VT-binding was detected within the infant glomerulus but not the adult. Binding of the toxin was removed when the section was pretreated with alpha-galactosidase, confirming the receptor-binding specificity for globotriaosyl ceramide (gal alpha 1-4gal beta 1-4 glucosylceramide), the glycolipid receptor for VT. These studies may suggest that differential localization of this glycolipid in the pediatric renal glomerulus is a risk factor for the development of HUS following infection with VTEC.

Alteration of the glycolipid binding specificity of the pig edema toxin from globotetraosyl to globotriaosyl ceramide alters in vivo tissue targetting and results in a verotoxin 1-like disease in pigs.

All members of the verotoxin (VT) family specifically recognize globo-series glycolipids on the surface of susceptible cells. Those toxins that are associated with human disease, VT1, VT2, and VT2c, bind to globotriaosyl ceramide (Gb3) while VT2e, which is associated with edema disease of swine, binds preferentially to globotetraosyl ceramide (Gb4). We were recently able to identify, using site-directed mutagenesis, amino acids in the binding subunit of these toxins that are important in defining their glycosphingolipid (GSL) binding specificity (Tyrrell, G. J., K. Ramotar, B. Boyd, B. W. Toye, C. A. Lingwood, and J. L. Brunton. 1992. Proc. Natl. Acad. Sci. USA. 89:524). The concomitant mutation of Gln64 and Lys66 in the VT2e binding subunit to the corresponding residues (Glu and Gln, respectively) found in VT2 effectively converted the GSL binding specificity of the mutant toxin from Gb4 to Gb3 in vitro. We now report that the altered carbohydrate recognition of the mutant toxin (termed GT3) has biological significance, resulting in a unique disease after intravascular injection into pigs as compared with classical VT2e-induced edema disease. The tissue localization of radiolabeled GT3 after intravascular injection was elevated in neural tissues compared with VT2e accumulation, while localization of GT3 to the gastrointestinal tract was relatively reduced. Accordingly, the pathological lesions after challenge with GT3 involved gross edema of the cerebrum, cerebellum, and brain stem, while purified VT2e caused hemorrhage and edema of the cerebellum, and submucosa of the stomach and large intestine. In addition, both radiolabeled toxins bound extensively to tissues not directly involved in the pathology of disease. VT2e, unlike GT3 or VT1, bound extensively to red cells, which have high levels of Gb4. The overall tissue distribution of VT2e was thus found to be influenced by regional blood flow to each organ and not solely by the Gb4 levels of these tissues. Conversely, the distribution of GT3 (and VT1), which cleared more rapidly from the circulation, correlated with respective tissue Gb3 levels rather than blood flow. These studies indicate the primary role of carbohydrate binding specificity in determining systemic pathology, suggest that the red cells act as a toxin carrier in edema disease, and indicate that red cell binding does not protect against the pathology of systemic verotoxemia.

Globotriaosyl Ceramide Glycolipid in Seminoma: Its Clinicopathological Importance in Differentiation from Testicular Malignant Lymphoma

Glycolipids were biochemically extracted from 14 specimens of seminoma, 2 of testicular malignant lymphoma (both of which were difficult to differentiate from seminoma with a high mitotic index) and 4 of normal testicle. The pattern of their expression was compared. Marked accumulation of globotriaosyl ceramide was observed in seminoma but it was present in a small amount in testicular malignant lymphoma. Differentiation between seminoma and malignant lymphoma is sometimes difficult by histopathological findings but it is considered to be greatly facilitated by examination of the pattern of glycolipid expression.

Chromatographic and Spectroscopic Analysis of Globotriaosyl Ceramide from Bovine Spermatozoa

Abstract The isolation and characterization of globotriaosyl ceramide from bovine spermatozoa is presented. Analysis of Gb3 by high-resolution proton nuclear magnetic resonance (1H-NMR), fast atom bombardment mass spectrometry (FAB-MS), mobility on HFTLC, and HPTLC-immunostaining; GLC/MS analysis of the partially methylated alditol acetates of the sugar units, and GLC analysis of the sphingoid bases and fatty acids present in the ceramide moietv. resulted in the characterization of mis major glycosphingolipid as Gal(αl-4)Gal(βl-4)Glc(β1-1)Cer.

Polyisobutylmethacrylate modifies glycolipid binding specificity of verotoxin 1 in thin-layer chromatogram overlay procedures

Verotoxins (or Shiga-like toxins) are a family of closely related toxins elaborated by Escherichia coli. At least three toxins have been described, VT1, VT2, and SLTII, in addition to Shiga toxin itself, and all bind to globotriaosyl ceramide, Gb 3. Some discrepancies exist in the literature regarding the binding of the toxins to Gb 4 as monitored by TLC overlay procedures. These procedures are widely used to investigate the specificity of carbohydrate-binding ligands. Polyisobutylmethacrylate, PIBM, is generally used in TLC overlay procedures to prevent silica loss and orient carbohydrate moieties for the binding of various ligands to glycolipids. We now report that pretreatment of chromatograms with PIBM modifies binding of VT1 to include Gb 4 and decreases binding to Gb 3 and the P 1 glycolipid. We suggest that PIBM can alter the conformation of the glycolipid oligosaccharide, and therefore caution is advised in analysis of ligand binding to glycolipids after treatment with this compound.

Antibodies Against Platelet Glycosphingolipids: Detection in Serum by Quantitative HPTLC-Autoradiography and Association With Autoimmune and Alloimmune Processes

AbstractIn order to assess the importance of glycosphingolipids (GSL) in the immunology of the platelet, serum antibody binding to immobilized, purified platelet GSLs have been quantitatively measured via high-performance thin-layer chromatography (HPTLC), 125l-radio-immunolabeling, autoradiography, and densitometry. Thirteen neutral GSL bands were detected at Rf .03 through .64 (CHCI3-CH3OH- H2O, 65:25:4) after extraction and chromatography (DEAE- Sephadex and Bio-sil A). Both IgM and IgG serum antibody binding was determined for 50 subjects from four groups: normal blood donors (NBD, n = 18); leukemia patients with nonimmune thrombocytopenia (NIT, n = 10); patients with systemic lupus erythematosus (SLE, n = 10); and patients with chronic autoimmune thrombocytopenia (CATP, n = 12). The ABO typing of these 50 subjects also allowed correlation of serum antibody binding with A blood group antigen expression. These studies reveal: (1) anti-GSL binding at band .06 is associated with blood group A alloantigen expression for both IgG and IgM (P <.0001) antibodies; (2) binding at bands .17, .27, and/or .46 is associated with general autoimmunity (SLE and CATP) for IgM (P < .0001); (3) binding at bands .35 and/or .38 is associated with platelet-specific autoimmunity (CATP) for IgG and/or IgM {P< .005); and (4) binding at bands .03, .20, .23, and/or .43 is frequently observed for sera from all groups. The platelet specificity of bands .35 and .38 was confirmed by comparative studies with human intestinal smooth muscle GSLs. Quantitation of the intensity of CATP-associated anti-GSL binding (86 ± 88 mm2) is comparable to anti-A alloantigen binding (57 ± 52 mm2). Two of the GSL bands associated with SLE and CATP appear to be the long-chain fatty-acyl forms of globotriaosyl ceramide (.27) and globotetraosyl ceramide (.17), which are the Pk and P blood group antigens, respectively. These studies indicate that neutral GSLs may be important antigens in both autoimmune and alloimmune processes involving the blood platelet.

Antibodies against platelet glycosphingolipids: detection in serum by quantitative HPTLC-autoradiography and association with autoimmune and alloimmune processes

In order to assess the importance of glycosphingolipids (GSL) in the immunology of the platelet, serum antibody binding to immobilized, purified platelet GSLs have been quantitatively measured via high- performance thin-layer chromatography (HPTLC), 125I-radio- immunolabeling, autoradiography, and densitometry. Thirteen neutral GSL bands were detected at Rf.03 through .64 (CHCl3-CH3OH-H2O, 65:25:4) after extraction and chromatography (DEAE-Sephadex and Bio-sil A). Both IgM and IgG serum antibody binding was determined for 50 subjects from four groups: normal blood donors (NBD, n = 18); leukemia patients with nonimmune thrombocytopenia (NIT, n = 10); patients with systemic lupus erythematosus (SLE, n = 10); and patients with chronic autoimmune thrombocytopenia (CATP, n = 12). The ABO typing of these 50 subjects also allowed correlation of serum antibody binding with A blood group antigen expression. These studies reveal: (1) anti-GSL binding at band .06 is associated with blood group A alloantigen expression for both IgG and IgM (P less than .0001) antibodies; (2) binding at bands .17, .27, and/or .46 is associated with general autoimmunity (SLE and CATP) for IgM (P less than .0001); (3) binding at bands .35 and/or .38 is associated with platelet-specific autoimmunity (CATP) for IgG and/or IgM (P less than .005); and (4) binding at bands .03, .20, .23, and/or .43 is frequently observed for sera from all groups. The platelet specificity of bands .35 and .38 was confirmed by comparative studies with human intestinal smooth muscle GSLs. Quantitation of the intensity of CATP-associated anti-GSL binding (86 +/- 88 mm2) is comparable to anti-A alloantigen binding (57 +/- 52 mm2). Two of the GSL bands associated with SLE and CATP appear to be the long-chain fatty-acyl forms of globotriaosyl ceramide (.27) and globotetraosyl ceramide (.17), which are the Pk and P blood group antigens, respectively. These studies indicate that neutral GSLs may be important antigens in both autoimmune and alloimmune processes involving the blood platelet.

Interaction of Shigella toxin with globotriaosyl ceramide receptor — Containing membranes: A fluorescence study

The interaction of the B-subunit of Shigella toxin with a globotriaosyl ceramide receptor incorporated into phosphatidylcholine vesicles was studied by fluorescence spectroscopy. From the position of the maximum in the emission spectrum and the accessibility to acrylamide quenching, it is concluded that a single trytophan of a free B-chain is located in a highly polar environment, most likely on the surface of the folded polypeptide chain. Binding of B-subunits to the membrane-associated globotriaosyl ceramide results in a strong enhancement of fluorescence intensity and a small blue-shift of the emission maximum; these effects suggest a conformational change in the protein which provides a new environment to a tryptophan residue. However, the polarity of this new environment is still relatively high — as indicated by the position of the emission maximum at 344 nm — and suggests that the receptor-bound B-chain remains largely on the membrane surface, without penetrating the hydrophobic interior of a lipid bilayer. On the other hand, the A-chains are shown to interact directly with the receptor-free lipid bilayers; this nonspecific interaction may play a role in the mechanism by which A-subunit traverses the membrane of a target cell.

Subpopulations of B lymphocytes in germinal centers.

With two new monoclonal antibodies and flow cytometry, we defined three subpopulations among B cells expressing binding sites for peanut agglutinin (i.e., B cells of the germinal center). On monoclonal antibody (5B5) binds globotriaosyl ceramide. The B lymphocytes binding 5B5 have binding sites for peanut agglutinin on the surface and express only small amounts of sIgD and sIgM. When tested against a panel of B cell lines, only Burkitt's lymphoma cells were 5B5+. Moreover, the 5B5+ cells have larger average sizes and a large fraction of proliferating cells. The other monoclonal antibody (HK23) binds a 90,000 protein. Lymphocytes binding HK23 are 5B5- and include T cells and a subpopulation of B cells. In contrast to 5B5+ cells, the HK23+ and peanut agglutinin positive B cells express a large amount of sIgM. These two subpopulations of germinal centers are distinct from the germinal center B cell subpopulation expressing the CD23 (Blast-2) antigen. The CD23+ B cells are 5B5- and express an intermediate level of HK23 antigen. In addition, CD23+ B cells are highly variable in number, whereas the proportions of HK23+ and 5B5+ cells are relatively stable.

Studies on the human blood group p system: An existence of UDP-Gal:Lactosylceramide α1→4 galactosyltransferase in the small p type cells

Human red blood cells and other tissues with the rare small p type lack all P antigens, and are assumed to be missing key glycosyltransferases in the synthetic pathway of P antigens. Galactosyltransferase activities of the P 1 and small p cell extracts were measured using lactosylceramide and GlcNAc as galactose acceptors. The two transferase activities of the small p lymphoblastoid cell extract were comparable to that of the P 1 cell extract. The anomeric configuration of the galactosylated lactosylceramide was established by digestion with α- and β-galactosidases, by identification of methylated products, and by staining with the monoclonal antibody against globotriaosyl ceramide (P k antigen). The results indicate that UDP-Gal:LacCer α1→4 Gal transferase, which produces the P k antigen from the precursor LacCer, exists in the small p cells. However, intact small p cells could not produce the P k antigen, and, instead, LacCer was accumulated in the cells. The P k enzyme appears to be not functional in the small p cells in vivo .