Isolation and structural characterization of glycosphingolipids of in vitro propagated human umbilical vein endothelial cells

To investigate in detail the expression of glycosphingolipids (GSLs) on endothelial cells, 4.85 x 10(9) human umbilical vein endothelial cells (HUVECs) were cultivated in a 2 l bioreactor using microcarriers as a support for anchorage dependent growing cells. Neutral GSLs and gangliosides were isolated and their structures were determined by TLC immunostaining, fast atom bombardment-mass spectrometry (FAB-MS) of the native GSLs, and gas chromatography-electron impact mass spectrometry (GC-EIMS) of partially methylated alditol acetates. GbOse4Cer, GbOse3Cer, and LacCer, all carrying mainly C24- and C16-fatty acid beside C18-sphingosine, were detected as the major neutral GSLs (36%, 23%, and 15% of the total orcinol stain, respectively); GlcCer, nLcOse4Cer, and nLcOse6Cer were expressed to substantial minor amounts (9%, 12%, and 5% of the total orcinol stain, respectively). TLC immunostaining revealed the presence of lipid bound Lewisx antigen, whereas the isomeric Lewisa structure was detectable only in very low quantities. GM3(Neu5Ac) with C18-sphingosine was the major ganglioside constituting about 90% of the whole ganglioside fraction. The fatty acid composition was determined by GC-MS of fatty acid methyl esters, indicating the predominance of C24- and C16-substituted GM3(Neu5Ac), followed by C18- and C22-substituted species. Terminally alpha2-3 sialylated neolacto-series ganglioside IV3Neu5Ac-nLcOse4Cer was the second most abundant ganglioside in HUVECs (8% of the total resorcinol stain), and IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer (together less than 2% of total resorcinol stain) were found in minor quantities. Lipid bound sialyl Lewisx antigen with poly-N-acetyllactosaminyl chains, and traces of gangliotetraose-type gangliosides GM1 and GD1a were identified by TLC immunostaining. The expression of dominant neutral GSLs LacCer, GbOse3Cer, and GbOse4Cer, and of ganglioside GM3(Neu5Ac) was assayed by indirect immunofluorescence microscopy of cell layers grown in chamber slides, each showing different plasma membrane and subcellular distribution patterns. The complete structural characterization of GSLs from HUVECs contributes to our understanding about their functional role, not only of the carbohydrate but also of the lipid moiety, as receptors for bacterial toxins, as cell surface antigens of cellular interaction and as receptors for blood components and macromolecules of the extracellular matrix.     

Appearance of a novel type of ganglioside (GD1 alpha) in a differentiation-resistant clone of mouse myeloid leukemia cells, M1-R1

Glycolipid compositions of three mouse myeloid leukemia cell clones, two that are sensitive to differentiation inducers (M1-T22 and M1-S1) and one that is differentiation-resistant (M1-R1), have been compared. The T22 and S1 clones contained glucosylceramide (GlcCer), lactosylceramide (LacCer) and gangliotriaosylceramide (Gg3Cer) as the major neutral glycolipids. The differentiation resistant clone, R1, was characterized by the appearance of globotriaosylceramide (Gb3Cer) and a decrease of Gg3Cer. There was a distinct difference in the ganglioside profile between the differentiation-inducible and -resistant clones: T22 and S1 cells contained no detectable amounts of ganglioside, whereas six different gangliosides were detected in the R1 clone. These gangliosides were isolated and identified as GM3, GM2, GM1a, GD1a, GM1b, and a unique disialoganglioside, GD1 alpha, having the following structure: (formula; see text) Based on these comparative studies, the relationship between the glycolipid composition and the differentiation potential of leukemia cells is discussed.     

Cell adhesion, spreading, and motility of GM3-expressing cells based on glycolipid-glycolipid interaction

Cell lines expressing varying levels of ganglioside GM3 at the cell surface show different degrees of adhesion and spreading on solid phase coated with such glycosphingolipids (GSLs) as Gg3 (GalNAc beta 1----4Gal beta 1----4Glc beta 1----1Cer), LacCer (Gal beta 1----4Glc beta 1----1Cer), or Gb4 (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer) (where Cer is ceramide), which may have structures complementary to GM3, but not on solid phase coated with various other GSLs. The degree of cell adhesion and spreading on Gg3 was correlated with the degree of cell-surface GM3 expression, as defined by reactivity with anti-GM3 monoclonal antibody (mAb) DH2. Only cells with high GM3 expression adhered on solid phase coated with LacCer or Gb4. Adhesion of GM3-expressing cells on Gg3-, LacCer-, and Gb4-coated solid phase is based on interaction of GM3 with Gg3 and, to a lesser extent, with LacCer and Gb4, as demonstrated by: (i) the interaction of the GM3 liposome with solid phase coated with Gg3, LacCer, and Gb4, respectively; (ii) the abolition of cell adhesion on each GSL-coated solid phase by treatment of cells with mAb DH2 or sialidase; and (iii) the inhibition of cell adhesion by treatment of GSL-coated solid phase with mAb specific to each GSL. Sialosyllactosyl-lysyllysine conjugate was bound to Gg3 adsorbed on a C18 silica gel column in the presence of bivalent cation, suggesting that the carbohydrate moiety of GM3 is involved in GM3-Gg3 interaction. Not only the adhesion and spreading of GM3-expressing cells, but also their cell motility was greatly enhanced on Gg3-coated solid phase, as determined by Transwell assay and phagokinetic track assay on a gold sol-coated surface. Spreading and motility of GM3-expressing cells on Gg3-coated solid phase were both inhibited by treatment of cells with mAb DH2 or sialidase. These results provide evidence that not only cell adhesion, but also spreading and motility in these cell lines are controlled by complementary GSL-GSL interaction.     

Glycosphingolipid-enriched signaling domain in mouse neuroblastoma Neuro2a cells. Mechanism of ganglioside-dependent neuritogenesis.

Differentiation and neuritogenesis of mouse neuroblastoma Neuro2a cells are induced by exogenous ganglioside but are not induced by nerve growth factor because its receptor is absent in these cells. In view of the emerging concept of the "glycosphingolipid-enriched domain" (GEM), we studied the mechanism of the ganglioside effect, focusing on the structure and function of such a domain. GEM in Neuro2a cells, separated as a low density membrane fraction, contains essentially all glycosphingolipids and sphingomyelin, together with five signal transducer molecules (c-Src, Lyn, Csk, Rho A, Ha-Ras). (3)H-Labeled Il(3)NeuAc-LacCer (GM3), Gb4Cer (globoside), and Il(3)NeuAc-Gg4Cer (GM1) added exogenously to cells were incorporated and concentrated in the low density GEM fraction. In contrast, more than 50% of glycerophospholipids and 30% of cholesterol were found in the high density fraction. (3)H-Labeled phosphatidylcholine added exogenously to cells was incorporated exclusively in the high density fraction. c-Src, the predominant signal transducer in the microdomain, was coimmunoprecipitated with anti-GM3 antibody DH2 or with anti-Csk; reciprocally, Csk was coimmunoprecipitated with anti-c-Src, indicating a close association of GM3, c-Src, and Csk. Brief stimulation of an isolated GEM fraction by the exogenous addition of GM3, but not lactosylceramide, caused enhanced c-Src phosphorylation with a concomitant decrease of Csk level in GEM. A decreased Csk/c-Src ratio in GEM may cause activation of c-Src because Csk is a negative regulator of c-Src. The effect of exogenous GM3 on c-Src activity was also observed in intact Neuro2a cells. Activation of c-Src was followed by rapid and prolonged (60 min) enhancement of mitogen-activated protein kinase activity leading to neuritogenesis. Thus, the ganglioside induction of neuritogenesis in Neuro2a cells is mediated by GEM structure and function.     

A new monoclonal antibody directed to sialyl alpha 2-3lactoneotetraosylceramide and its application for detection of human gastrointestinal neoplasms

A new monoclonal antibody (NS24) directed to the N-acetylneuraminyl alpha 2-3Gal beta 1-4GlcNAc residue in type II sugar chain of N-acetylneuraminyllactoneotetraosylceramide [sialylparagloboside, IV3(NeuAc)nLc4Cer] was prepared by hybridoma technique. Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, IV3(NeuAc)nLc4Cer, and lipopolysaccharides from Salmonella minnesota R595 were used for immunization with IV3(NeuAc)nLc4Cer isolated from human erythrocytes. This method allowed the fusion of spleen cells of immunized mouse with myeloma cells only three days after immunization. NS24 reacted specifically to both naturally occurring and chemically synthesized IV3-(NeuAc)nLc4Cer, whereas it has no reactivity to structurally related gangliosides, such as IV6(NeuAc)nLc4Cer, N-glycolylneuraminyl alpha 2-3lactoneotetraosylceramide [IV3(NeuGc)-nLc4Cer], i-active ganglioside [VI3(NeuAc)nLc6Cer], I-active ganglioside [VIII3(NeuAc)-VI3(NeuAc)IV6kladoLc8Cer], GM4(NeuAc), GM3(NeuAc), GM3(NeuGc), GM1b(NeuAc), GD3-(NeuAc), other ganglio-series gangliosides, sulfatide, and paragloboside (nLc4Cer). Synthetic N-acetylneuraminyl alpha 2-3lactotetraosylceramide [IV3(NeuAc)Lc4Cer] and its asialo-derivative (Lc4Cer) carrying type I sugar chain also showed no reaction with NS24. One to 100 pmol of IV3(NeuAc)nLc4Cer was detected dose-dependently by a thin-layer chromatography/enzyme immunostaining procedure. Human gastric carcinomas showed positive reactions with NS24 immunochemically and histochemically. NS24 reacted preferentially with poorly differentiated adenocarcinomas rather than well differentiated ones.     

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

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

Defective Pericyte Recruitment of Villous Stromal Vessels as the Possible Etiologic Cause of Hydropic Change in Complete Hydatidiform Mole

The pathogenetic mechanism underlying the hydropic change in complete hydatidiform moles (CHMs) is poorly understood. A growing body of data suggests that pericytes play a role in vascular maturation. Since maturation of villous stromal vessels in CHMs is markedly impaired at early stages, we postulated that a defect in pericytes around stromal vessels in chorionic villi might cause vascular immaturity and subsequent hydropic change. To investigate this, we examined several markers of pericytes, namely, α-smooth muscle actin (α-SMA), platelet-derived growth factor receptor-β (PDGFR-β), and desmin, in 61 normally developing placentas and 41 CHMs with gestational ages of 4–12 weeks. The ultrastructure of villous stromal vessels was also examined. Mature blood vessels from normal placentas show patent vascular lumens and formed hematopoietic components in the villous stroma. α-SMA and PDGFR-β expression in the villous stroma gradually increased and extended from the chorionic plate to peripheral villous branches. The labeled cells formed a reticular network in the villous stroma and, after week 7, encircled villous stromal vessels. In comparison, α-SMA and PDGFR-β expression in the villous stroma and stromal vessels of CHMs was significantly lower (p<0.05). Ultrastructurally, endothelial cells in villous stromal vessels in normal placentas were consistently attached by pericytes after week 7 when the vessels formed distinct lumen, whereas the villous stromal vessels in CHMs consisted of linear chains of endothelial cells, often disclosing primitive clefts without hematopoietic cells inside, and neither pericytes nor basal lamina surrounded the endothelial cells at any gestational age studied. This suggests that pericytes recruitment around villous stromal vessels is defective in CHMs and links to the persistent vascular immaturity of the villous stroma in CHMs, which in turns leads to hydropic villi.     

Expression of G-Protein Subunit α-14 Is Increased in Human Placentas from Preeclamptic Pregnancies

G-proteins mediate cellular function upon interaction with G-protein coupled receptors. Of the 16 mammalian G-protein α subunits identified, G-protein subunit α-11 (GNA11) and -14 (GNA14) have been implicated in modulating hypertension and endothelial function. However, little is known about their expression and roles in human placentas. Here, we examined GNA11 and GNA14 protein expression in first trimester (FT), normal term (NT), and severe preeclamptic (sPE) human placentas as well as in NT human umbilical cords. We found that GNA11 and GNA14 were immunolocalized primarily in trophoblasts, villous stromal cells, and endothelial cells in placentas as well as in endothelial and/or smooth muscle cells of the umbilical cord artery and vein. Western blotting revealed that the GNA14, but not GNA11, protein levels were increased (2.5-2.9 fold; p<0.01) in sPE vs. NT placentas. GNA11 protein was detected only in NT, but not FT, placentas, whereas GNA14 protein levels were increased (7.7-10.6 fold; p<0.01) in NT vs. FT placentas. Thus, GNA11 and GNA14 may mediate the function of several cell types in placentas. Moreover, the high expression of GNA14 in sPE placentas may also imply its importance in sPE pregnancies as in the other hypertension-related disorders.     

Immunohistological and flow cytometric analysis of glycosphingolipid expression in mouse lymphoid tissues.

Expression of neutral glycosphingolipids (GSLs) and gangliosides in normal lymphoid tissues and cells has been studied mostly by biochemical and immunochemical analysis of lipid extracts separated by thin-layer chromatography. GSLs and gangliosides involved in the GM1b biosynthetic pathway were assigned to T-lymphocytes, whereas B-cell gangliosides and GSLs have been poorly characterized in former publications. We used specific polyclonal antibodies in immunohistochemistry and flow cytometry to analyze the distribution of globotriaosylceramide (Gb(3)Cer), globoside (Gb(4)Cer), gangliotriaosylceramide (Gg(3)Cer), gangliotetraosylceramide (Gg(4)Cer), and gangliosides GM3 and GalNAc-GM1b in the mouse thymus, spleen, and lymph node. Immature thymocytes expressed epitopes recognized by all antibodies, except for anti-Gb(4)Cer. Mature thymocytes bound only antibodies to GalNAc-GM1b, Gg(4)Cer, and Gb(4)Cer. In secondary lymphoid organs, antibodies to globo-series GSLs bound to vascular spaces of secondary lymphoid organs, whereas the ganglio-series GSL antibodies recognized lymphocyte-containing regions. In a Western blotting analysis, only GalNAc-GM1b antibody recognized a specific protein band in all three organs. Flow cytometric analysis of spleen and lymph node cells revealed that B-cells carried epitopes recognized by all antibodies, whereas the T-cell GSL repertoire was mostly oriented to ganglio-series-neutral GSLs and GM1b-type gangliosides. The results of immunohistochemistry and flow cytometry were not always identical, possibly because of crossreactivity to glycoprotein-linked oligosaccharides and/or differences between cell surface carbohydrate profiles of isolated cells and cells in a tissue environment.     

Preeclampsia Does Not Alter Vascular Growth and Expression of CD31 and Vascular Endothelial Cadherin in Human Placentas

Preeclampsia is characterized by maternal endothelial dysfunction (e.g., increased maternal vascular permeability caused by the disassembly of endothelial junction proteins). However, it is unclear if preeclampsia is associated with impaired vascular growth and expression of endothelial junction proteins in human placentas. Herein, we examined vascular growth in placentas from women with normal term (NT) and preeclamptic (PE) pregnancies using two endothelial junction proteins as endothelial markers: CD31 and vascular endothelial-cadherin (VE-Cad). We also compared protein and mRNA expression of CD31 and VE-Cad between NT and PE placentas, and determined the alternatively spliced expression of CD31 using PCR. We found that CD31 and VE-Cad were immunolocalized predominantly in villous endothelial cells. However, capillary number density (total capillary number per unit villous area) and capillary area density (total capillary lumen area per unit villous area) as well as CD31 and VE-Cad protein and mRNA levels were similar between NT and PE placentas. PCR in combination with sequence analysis revealed a single, full-length CD31, suggesting that there are no alternatively spliced isoform of CD31 expressed in placentas. These data indicate that preeclampsia does not significantly affect vascular growth or the expression of endothelial junction proteins in human placentas.     

Association of glycosphingolipids with intermediate filaments of mesenchymal, epithelial, glial, and muscle cells.

We reported recently that two glycosphingolipids (GSLs), globoside (Gb4) and ganglioside GM3, colocalized with vimentin intermediate filaments of human umbilical vein endothelial cells. To determine whether this association is unique to endothelial cells or to vimentin, we analyzed a variety of cell types. Double-label immunofluorescent staining of fixed, permeabilized cells, with and without colcemid treatment, was performed with antibodies against glycolipids and intermediate filaments. Globoside colocalized with vimentin in human and mouse fibroblasts, with desmin in smooth muscle cells, with keratin in keratinocytes and hepatoma cells, and with glial fibrillary acidic protein (GFAP) in glial cells. Globoside colocalization was detected only with vimentin in MDCK and HeLa cells, which contain separate vimentin and keratin networks. GM3 ganglioside also colocalized with vimentin in human fibroblasts. Association of other GSLs with intermediate filaments was not detected by immunofluorescence, but all cell GSLs were detected in cytoskeletal fractions of metabolically labelled endothelial cells. These observations indicate that globoside colocalizes with vimentin, desmin, kertain and GFAP, with a preference for vimentin in cells that contain both vimentin and keratin networks. The nature of the association is not yet known. Globoside and GM3 may be present in vesicles associated with intermediate filaments (IF), or bound directly to IF or IF associated proteins. The prevalence of this association suggests that colocalization of globoside with the intermediate filament network has functional significance. We are investigating the possibility that intermediate filaments participate in the intracellular transport and sorting of glycosphingolipids.     

Activation of ganglioside GM3 biosynthesis in human blood mononuclear cells in atherosclerosis

Using blood monocytes and lymphocytes from atherosclerotic patients and healthy subjects we have investigated a role of ganglioside GM3 in monocyte adhesion to cultured human umbilical vein endothelial cells (HUVEC). The results showed that activity of GM3 synthase and cellular levels of ganglioside GM3 in blood mononuclear cells from atherosclerotic patients were several-fold higher than those from healthy subjects. In monocytes the activity of GM3 synthase was one order of magnitude higher than in lymphocytes from both groups studied; this suggests the major contribution of monocytes to enhanced biosynthesis and levels of GM3 in mononuclear cells in atherosclerosis. Enrichment of monocytes from healthy subjects with ganglioside GM3 by their incubation in the medium containing this ganglioside increased adhesion of these monocytes to HUVEC up to the level typical for monocytes from atherosclerotic patients. In addition, an increase in CD11b integrin expression comparable to that seen in lipopolysaccharide-activated monocytes was observed. It is suggested that in atherosclerosis the enhanced cellular levels of GM3 in monocytes and lymphocytes may be an important element of cell activation that facilitates their adhesion to endothelial cells and penetration into intima.     

Glycosphingolipid composition of a renal cell line (MDCK) and its ouabain-resistant mutant

Glycosphingolipids were isolated from a canine kidney cell line (MDCK) and its ouabain-resistant mutant (MDCK-OR) by solvent extraction, mild alkaline methanolysis, a DEAE-Sephadex column, and preparative TLC. The glycolipids were characterized by their mobilities on TLC, an analysis of carbohydrates as trimethylsilyl methyl glycosides and acetates of partially methylated alditols, as well as by treatment with specific glycosidases. In the neutral glycolipid fraction of both cell lines, galactosylceramide (GalCer), glucosylceramide (GlcCer), lactosylceramide (LacCer), digalactosylceramide (Ga2Cer), globotriaosylceramide (Gb3Cer), globoside (Gb4Cer), and the Forssman antigen (IV3GalNAc alpha-Gb4Cer) were identified. The contents of Ga2Cer (4.4 nmol/mg protein), Gb3Cer (0.6), Gb4Cer (2.9), and IV3GalNac alpha-Gb4Cer (19.5) in MDCK-OR were 1.4- to 2.1-fold higher than those in MDCK, while the concentrations of GlcCer (5.3) and LacCer (1.4) in MDCK-OR were about half of those in MDCK. Among acidic glycolipids of MDCK-OR, galactosyl sulfatide (GalCer-I3-sulfate) and lactosyl sulfatide (LacCer-II3-sulfate) were increased to 1.9 (2.7-fold) and 0.2 nmol/mg protein (2.0-fold), respectively, as compared to MDCK. However, N-acetylneuraminosyllactosylceramide (GM3), the predominant ganglioside in both cell lines, was decreased to about one third of the level (1.5 nmol/mg protein) in the parent MDCK (4.7 nmol/mg protein). The fatty acid of the glycolipids in both cell lines consisted mainly of saturated acids of 16, 18, 22, and 24 carbons.     

Establishment of a monoclonal antibody directed against Gb3Cer/CD77: a useful immunochemical reagent for a differentiation marker in Burkitt's Iymphoma and germinal centre B cells

A new monoclonal antibody (TU-1) directed against the Galα1-4Galβ1-4Glc residue of the Gb3Cer/CD77 antigen was prepared by the hybridoma technique following immunization of mice with an emulsion composed of monophosphoryl lipid A, trehalose dimycolate, and Gb3Cer isolated from porcine erythrocytes. TU-1 showed reactivity towards Gb3Cer and lyso-Gb3Cer (Galα1-4Galβ1-4Glcβ1-1′Sph), although the reactivity towards lyso-Gb3Cer was about 10-fold lower than that to Gb3Cer. But it did not react with other structurally-related glycolipids, such as LacCer (Galβ1-4Glcβ1-1′Cer), Gg3Cer, Gg4Cer, Gb4Cer (GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1′Cer), galactosylparagloboside (Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer), sulfatide (HSO3-3Galβ1-1′Cer), other gangliosides (GM3, GM2, GM1a, GD1a and GT1b), or P1 antigen (Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer) among neutral glycolipids prepared from P1 phenotype red blood cells. Furthermore, TU-1 reacted with viable lymphoma cells, such as human Burkitt lymphoma cell line, Daudi, and Epstein-Barr virus (EBV)-transformed B cells by the immunofluorescence method, and also with germinal centre B cells in human tonsil and vessel endothelial cells in human thymus histochemically. These results indicate that TU-1 is a monoclonal antibody directed against Gb3Cer/CD77 antigen and can be utilized as a diagnostic reagent for Burkitt's lymphoma and also for detection of the blood group Pk antigen in glycolipid extracts of erythrocytes. Abbreviations: ATL, adult T-cell leukaemia; BSA, bovine serum albumin; Cer, ceramide; DPPC, L-α-dipalmitoylphosphatidylcholine; EBV, Epstein-Barr virus; FCS, fetal calf serum; GalCer, Galβ1-1′Cer; GlcCer, Glcβ1-1′Cer; LacCer, Galβ1-4Glcβ1-1′Cer; Gb3Cer, Galα1-4Galβ1-4Glcβ1-1′Cer; Iyso-Gb3Cer, Galα1-4Galβ1-4Glc1-1′Sph; Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glc1-1′Cer; galactosylparagloboside, Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; Gg3Cer, GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; Gg4Cer, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; GM3, Neu5Acα2-3Galβ1-4Glcβ1-1′Cer; GM2, GalNAcβ1-4(Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; GM1a, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1a, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1b, Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GT1b, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; MAb, monoclonal antibody; MPL, monophosphoryl lipid A; P1 antigen, Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; PVP, polyvinylpyrolidone; Sph, sphingosine; sulfatide, HSO3-Galβ1-1′Cer; TDM, trehalose dimycolate; TLC, thin-layer chromatography This revised version was published online in November 2006 with corrections to the Cover Date.     

A new ganglioside showing choleragenoid-binding activity in mouse spleen

A new ganglioside showing choleragenoid-binding activity was purified from mouse spleen and characterized. From the results of sugar-composition analysis, enzymatic hydrolysis, a permethylation study, 1H-NMR spectroscopy, and negative-ion fast atom bombardment mass spectrometry, the structure of the ganglioside was determined to be as follows: Gal beta 1-3GalNAc beta 1-4Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1'ceramide 3----NeuGc alpha 2 This ganglioside contains a terminal tetrasaccharide structure identical with that of II3NeuGc alpha-Gg4Cer (GM1(NeuGc]. By means of a TLC-immunobinding assay and an enzyme-linked immunosorbent assay, the ganglioside was demonstrated to have almost the same choleragenoid-binding activity as GM1. Another ganglioside, that migrated faster than the new choleragenoid-binding ganglioside, was also purified from the same source material and identified as IV4GalNAc beta,IV3NeuGc alpha-Gg4Cer (GalNAc-GM1b(NeuGc]. Since, in the previous study, we demonstrated the existence of IV3NeuGc alpha-Gg4Cer (GM1b(NeuGc] in mouse spleen (Nakamura, K. et al. (1984) J. Biochem. 96, 949-957), the results of this study suggest that the new choleragenoid-binding ganglioside is synthesized from GM1b(NeuGc) through GalNAc-GM1b(NeuGc).     

Gangliosides of murine T lymphocyte subpopulations

Gangliosides from murine T lymphoblasts were analyzed by high-performance thin-layer chromatography followed by in situ neuraminidase treatment and immunostaining of the resulting asialogangliosides and compared with those from thymocytes and cloned T lymphocytes with defined functions. The ganglioside IVNeuGc/Ac-GgOse5Cer (GalNAc-GM1b), a marker for T lymphoblasts [Müthing, J., Egge, H., Kniep, B., & Mühlradt, P. F. (1987) Eur. J. Biochem. 163, 407-416], was found only in small amounts as the N-acetylated species in gangliosides from thymocytes and a cytolytic T cell clone. Two helper clones expressed this ganglioside like T blasts. The structures of the two major disialogangliosides from T blasts, IVNeuAc,IIINeuAc-GgOse4Cer (GD1 alpha type) with C24:0/24:1 and C16:0 fatty acids, were elucidated by neuraminidase treatment and immunostaining and by fast atom bombardment mass spectrometry. Gangliosides of this type were detected in thymocytes only in minor amounts, whereas GM1b-type gangliosides prevailed in cells from this organ. Analysis of the T lymphoblast gangliosides from six genetically unrelated mouse strains showed that terminally sialylated GgOse4Cer (GM1b), IVNeuAc-GgOse5Cer (GalNAc-GM1b), and IVNeuAc,IIINeuAc-GgOse4Cer (GD1 alpha) were conserved structures in all strains examined. We conclude that maturation or stimulation of T cells may be correlated with elongation of a common GM1b-type precursor structure resulting in GalNAc-GM1b or GD1 alpha-type gangliosides.     

Glycosphingolipid biosynthesis during myogenesis of rat L6 cells in vitro

Summary Glycosphingolipid biosynthesis was examined using [³H]-galactose as a precursor as rat L6 myoblasts fused to form multinucleated myotubes. Incorporation of label into neutral glycolipids decreased steadily as the population of myotubes increased, so that final biosynthesis was one-half that observed with myoblasts (p < 0.02). Conversely, ganglioside biosynthesis doubled during myoblast confluency (p < 0.02) and then decreased as myotubes formed. Qualitatively, L6 cells synthesized large amounts of ganglioside GM3 during all myogenic phases. The major neutral glycosphingolipid products were lactosylceramide and paragloboside (nLcOse₄Cer). Few changes in TLC autoradiographic patterns were noted during differentiation, with the exception of a slight decrease in ganglioside GM1. The results indicate that the biosynthesis of glycosphingolipids is tightly regulated during myogenesis in vitro and suggest a role for membrane gangliosides in muscle cell differentiation.Abbreviations GM1 II³NeuAc-GgOse₄Cer - GM3 II³NeuAc-GgOse²Cer - MG4 IV³NeuAc-nLcOse₄Cer - MG6 VI³NeuAc V⁴Gal-IV³GlcNAc-nLcOse⁴Cer - TLC Thin-Layer Chromatography - DMEM Dulbecco's Modified Eagles' Medium     

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

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