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.     

Monoclonal antibody detects monosialogangliosides having a sialic acid alpha 2----3-galactosyl residue

A murine monoclonal antibody (mAb), designated mAb 202, was generated using a human melanoma cell line, UCLASO-M14 as the immunogen. mAb 202 reacted with two (GM2 and GM3) of the four (GM2, GM3, GD2, and GD3) gangliosides expressed by M14. Several authentic monosialogangliosides, including GM4, GM3, GM2, GM1, GM1b, and sialylparagloboside were then tested for their binding to 202 mAb by the immune adherence inhibition assay, TLC-enzyme immunostaining, and enzyme-linked immunosorbent assay. All showed positive binding but in varying degrees. GM4 showed the strongest affinity. No significant differences of reactivity were observed between the sialic acid derivatives, N-acetyl and N-glycolyl, in these gangliosides. Disialogangliosides such as GD3, GD2, GD1a, and GD1b, trisialoganglioside GT1b, and neutral glycolipids including GlcCer, GalCer, LacCer, GbOs3Cer, GbOs4Cer, GgOs3Cer, GgOs4Cer, and nLcOs4Cer were all negative. These results indicate that the 202 mAb detects sialyl alpha 2----3Gal residue in the monosialoganglioside, irrespective of the internal structure. Since GM4 is not expressed by M14 cells, the terminal disaccharide (sialyl alpha 2----3Gal) in GM3 and/or GM2 must have been the epitope responsible for the generation of the antibody.     

Expression machinery of GM4: the excess amounts of GM3/GM4S synthase (ST3GAL5) are necessary for GM4 synthesis in mammalian cells

The ganglioside GM4 is a sialic acid-containing glycosphingolipid mainly expressed in mammalian brain and erythrocytes. GM4 is synthesized by the sialylation of galactosylceramide (GalCer), while the ganglioside GM3 is synthesized by the sialylation of lactosylceramide (LacCer). Recently, the enzyme GM3 synthase was found to be responsible for the synthesis of GM4 in vitro and in vivo, yet the mechanism behind GM4 expression in cells remains unclear. In this study, we attempted to establish GM4-reconstituted cells to reveal the regulation of GM4 synthesis. Interestingly, GM4 was not detected in RPMI 1846 cells expressing LacCer, GalCer, and GM3. Similarly, GM4 was not detected in CHO-K1 cells, even when such cells expressing LacCer and GM3 were stably transfected with the GalCer synthase (GalCerS) gene. GM4 became detectable only when the GM3/GM4 synthase (GM3/GM4S, ST3GAL5) gene was overexpressed in either RPMI 1846 or CHO-K1/GalCerS cells. A mutant of the B16 melanoma cell line, GM-95, lacks GlcCer and LacCer, due to an absence of GlcCer synthase, but carries endogenous LacCer synthase and GM3/GM4S. GalCer became detectable after transfection of GalCerS into GM95 cells, but the GM95/GalCerS reconstituted cells did not express GM4, indicating that competition between the substrates LacCer and GalCer for GM3/GM4S does not cause the failure of GM4 synthesis. These results suggest that the expression machinery of GM4 under physiological conditions is independent from that of GM3.     

GM3 ganglioside as melanoma specific antigen and its biological function

We have shown that a syngenic monoclonal antibody, M2590, established after immunization of C57BL/6 mice with B16 melanoma cells, recognized GM3 (NeuAc) ganglioside. Although GM3 is widely distributed among various normal cells and tissues, the antibody did not react with them. However, it reacted exclusively with melanoma cells from mouse, hamster and human. Preliminary experiments suggested that proteins and lipids as well as GM3 density on B16 cells are involved in the reactivity of GM3 with the antibody. Then, we investigated the biological function of the melanoma antigen, which was secreted from B16 cells into the culture medium. This soluble antigen was shown to suppress the positive immune responses by inhibiting CTL activity in the effector phase and by induction of specific suppressor T cells (Ts) that block CTL generation in the induction phase. Liposomes containing GM3 (NeuAc) but not GM3 (NeuGc) can effectively induce the melanoma specific Ts as did the soluble antigen. The results indicated the tumor cells can escape from host-immune system by stimulating the repertoire of Ts for self-antigen, GM3. To understand the biological role of GM3, we have established mutant clones of no-expressor of GM3 recognized by M2590. The clones were found to have lower attachment to laminin and type IV collagen and poor ability of lung metastasis.     

Mouse monoclonal antibodies with specificity for the melanoma-associated ganglioside disialyllactosylceramide (GD3) also react with the structural analogue disialylparagloboside

A mouse monoclonal IgM antibody, 4.2, has previously been shown to bind preferentially to the surface of human malignant melanoma cells and to have specificity for the GD3 ganglioside (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4GlcCer). Using overlay of antibodies on thin-layer chromatograms with glycolipids of various sources, it was shown that antibody 4.2, a further IgM and two IgG3 mouse monoclonal antibodies, selected on the basis of reactivity with GD3, also bound with similar strength to the structural analogue NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4GlcNac beta 1----3Gal beta 1----4GlcCer or disialylparagloboside. The SK-MEL 28 melanoma cell line used for immunization was shown to contain a large amount of GD3 but to lack disialylparagloboside. The demonstrated cross-reactivity may be of importance when considering the use of these antibodies for biochemical and medical purposes.     

Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis

Glycosphingolipids are controlled by the spatial organization of their metabolism and by transport specificity. Using immunoelectron microscopy, we localize to the Golgi stack the glycosyltransferases that produce glucosylceramide (GlcCer), lactosylceramide (LacCer), and GM3. GlcCer is synthesized on the cytosolic side and must translocate across to the Golgi lumen for LacCer synthesis. However, only very little natural GlcCer translocates across the Golgi in vitro. As GlcCer reaches the cell surface when Golgi vesicular trafficking is inhibited, it must translocate across a post-Golgi membrane. Concanamycin, a vacuolar proton pump inhibitor, blocks translocation independently of multidrug transporters that are known to translocate short-chain GlcCer. Concanamycin did not reduce LacCer and GM3 synthesis. Thus, GlcCer destined for glycolipid synthesis follows a different pathway and transports back into the endoplasmic reticulum (ER) via the late Golgi protein FAPP2. FAPP2 knockdown strongly reduces GM3 synthesis. Overall, we show that newly synthesized GlcCer enters two pathways: one toward the noncytosolic surface of a post-Golgi membrane and one via the ER toward the Golgi lumen LacCer synthase.     

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis^x (fucosylneolactonorpentaosyl ceramide; Le^x), difucosylneolactonorhexaosyl ceramide (dimeric Le^x), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le^x and the complex type of sialyl-Le^x derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le^x glycolipids and sialyl-Le^x were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le^x glycolipid was located on the tip of fine cellular processes. The unique localization of the Le^x glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.     

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.     

Cell type specific localization of sphingomyelin biosynthesis

We have studied the incorporation of [(14)C]serine and of [(3)H]sphingosine into sphingomyelin in the presence or absence of brefeldin A (BFA) in three different cell types. Administration of BFA (1 microgram/ml) to fibroblasts for 24 h increased the incorporation of label into sphingomyelin 1.5-3 fold compared with untreated controls. In contrast, BFA strongly decreased sphingomyelin biosynthesis (4-5 fold) in cerebellar neurons as well as in neuroblastoma cells. The effect of BFA on glycosphingolipid formation, however, was similar in all three cell types studied: an increased labeling of the precursor glycolipids GlcCer, LacCer, GM3 and GD3 was paralleled by a decreased formation of complex gangliosides, GM1, GD1a, GT1b and GQ1b. Our data therefore suggest that in neuronal cells sphingomyelin synthesis, like the formation of complex gangliosides, is localized primarily distal to the BFA block, in a post-Golgi compartment, most probably the trans-Golgi network, whereas in fibroblasts sphingomyelin biosynthesis is mainly localized prior to the BFA block, in the Golgi apparatus, as has been shown for LacCer, GlcCer, GM3 and GD3 synthases.     

Density-dependent recognition of cell surface GM3 by a certain anti-melanoma antibody, and GM3 lactone as a possible immunogen: requirements for tumor-associated antigen and immunogen

A mouse monoclonal antibody M2590, previously established after immunization of mice (C57BL/6) with syngeneic melanoma B16 cells and showing preferential reactivity with various types of melanoma over other tumor and normal cells or tissues, was shown to be directed to GM3 ganglioside. Since GM3 is widely distributed in essentially all types of animal cells, there is a conflict with the concept of a tumor-associated antigen and immunogen. Studies on the reactivity of M2590 antibody with various cells having different GM3 density at their cell surface, including cells treated with sialidase, liposomes, and solid-phase lipid layer containing different GM3 concentrations, have indicated that 1) reactivity of the antibody M2590 depends greatly on the density of GM3 exposed at the cell surface, on liposomes, or on solid phase; and 2) there is a threshold density that is recognized by the antibody in all-or-none fashion. In addition, the antibody M2590 reacts not only with GM3 but also with GM3 lactone, and the binding affinity of the antibody to GM3 lactone is strikingly higher than to GM3; however, the antibody does not react with GM3 ethyl ester. GM3 lactone was detected in melanoma as 3H-labeled GM3 gangliosidol after melanoma cells were directly treated with NaB[3H]4. A comparative immunization of BALB/c mice with GM3 and GM3 lactone showed that GM3 lactone is a much stronger immunogen than GM3, although the antibody elicited reacts with both GM3 and its lactone. Thus, the real immunogen could be GM3 lactone, although it is a minor membrane component.     

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.     

Analysis of gangliosides from carp intestinal mucosa

The gangliosides of carp intestinal mucosa were isolated and analysed by thin-layer chromatography (TLC), TLC immunostaining test, and TLC/secondary ion mass spectrometry (TLC/SIMS). Four species of gangliosides, designated as G-1, G-2, G-3 and G-4, were separated on TLC. The TLC/SIMS analysis of the G-1 ganglioside of carp intestinal mucosa revealed a series of [M-H](-)ions from m/z 1061 to m/z 1131 representing the molecular mass range of GM4-like ganglioside with NeuAc. G-2, G-3 and G-4 gangliosides were analysed by the TLC immunostaining test. G-2 ganglioside was recognised by the monoclonal antibody specific for ganglioside GM1 (AGM-1 monoclonal antibody). However, G-3 ganglioside migrating on TLC between GM3 and GM1 ganglioside was not recognised by anti-GM3 monoclonal antibody and by AGM-1 monoclonal antibody. Furthermore, G-4 ganglioside with a similar TLC mobility as GD1a ganglioside did not show the reactivity to the anti-GD1a monoclonal antibody. In addition using the AGM-1 monoclonal antibody, the expression of GM1 ganglioside in the carp intestinal tissue was studied. GM1 ganglioside was detected on the epithelial cell surface of carp intestinal mucosa.     

A novel ganglioside, de-N-acetyl-GM3 (II3NeuNH2LacCer), acting as a strong promoter for epidermal growth factor receptor kinase and as a stimulator for cell growth

A novel ganglioside, de-N-acetyl-GM3 (neuraminyllactosylceramide, II3NeuNH2LacCer), was found in the monosialoganglioside fraction of A431 cells and B16 melanoma cells by high-performance liquid chromatography, thin-layer chromatography, and immunoblotting with its specific monoclonal antibody DH5. This novel type of membrane ganglioside strongly enhanced the kinase activity associated with the epidermal growth factor (EGF) receptor, and it showed 32, 35, and 12% growth stimulation as compared with control cultures of A431, Swiss 3T3, and B16 melanoma cells, respectively. Exogenously added de-N-acetyl-GM3 did not alter the affinity of EGF binding to its receptor. These properties of de-N-acetyl-GM3 are in striking contrast to those of GM3 and its lyso derivative (lyso-GM3) which were previously shown to inhibit EGF receptor kinase activity and to inhibit growth in the same cells. These data indicate that de-N-acetylation at the sialic acid moiety of GM3 ganglioside is an important mechanism for modulation of EGF-dependent cell growth. The mechanism is antagonistic to that of GM3-dependent modulation of receptor function.     

Glucosylceramide Synthesized In Vitro from Endogenous Ceramide is Uncoupled from Synthesis of Lactosylceramide in Golgi Membranes from Chicken Embryo Neural Retina Cells

It is known that ceramide (Cer), the precursor of sphingoglycolipids and of sphingomyelin, participates in events leading to activation of the apoptotic pathway, and per se or through conversion to glucosylceramide (GlcCer) modulates formation of neuritic processes in developing neurons. To learn about the fate of de novo synthesized Cer and GlcCer we examined, in Golgi membranes from chicken embryo neural retina cells, the metabolic relationships of endogenous Cer, GlcCer and lactosylceramide (LacCer). Incubation of the membranes with UDP-[³H]Glc revealed a pool of endogenous Cer useful for synthesis of GlcCer. Most of the GlcCer synthesized, however, was not used for synthesis of LacCer, indicating that it was functionally uncoupled from LacCer synthase. On the other hand, incubation with UDP-[³H]Gal revealed a pool of endogenous GlcCer that depending of the integrity of the membranes was functionally coupled to LacCer and ganglioside synthesis. These results indicate that most GlcCer formed in vitro from Cer is topologically segregated from the synthesis of LacCer. However, subfractionation in sucrose gradients of Golgi membranes labeled with both precursors failed to separate membranes enriched in [³H]GlcCer from those enriched in [³H]Gal-labeled LacCer. It is concluded that despite both transfer steps co-localize in the Golgi membranes, coupling of GlcCer synthesis to LacCer synthesis requires conditions not present in our in vitro assay. This suggests that a coupling activity exists that could be relevant for regulation of the cytoplasmic levels of Cer and GlcCer.     

Inhibitory effects of gangliosides on immune reactions of antibodies to neutral glycolipids in liposomes

Specific immune damage to liposomes containing Forssman or globoside glycolipid was inhibited when the liposomes also contained ganglioside. The activity of a human monoclonal Waldenstr?m macroglobulin antibody to Forssman glycolipid was inhibited by each of three gangliosides tested, GM3, GD1a and GD1b. Inhibition of the monoclonal antibody was dependent on the amount of ganglioside in the liposomes, and was diminished by reducing the relative amount of ganglioside. Inhibition also correlated positively with the number of ganglioside sialic acid groups, with inhibition by GT1b greater than GD1a greater than GM3. Naturally occurring human antibodies to globoside glycolipid were detected in 18% (9 out of 50) of normal human sera tested. Immune damage to liposomes induced by each of the three highest-reacting human anti-globoside sera was blocked by liposomal GM3. We conclude that gangliosides can strongly influence immune damage to membranes induced by antibody interactions with adjacent neutral glycolipids.     

High-performance liquid chromatography-mass spectrometry of glycosphingolipids: II. Application to neutral glycolipids and monosialogangliosides

We previously reported a method of high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC/FAB/MS) for the structural characterization of molecular species of GlcCer and IV3 beta Gal-Gb4Cer [M. Suzuki et al. (1989) J. Biochem. 105, 829-833]. In this paper, we report a modification of this HPLC/FAB/MS method, which was used for the separation and characterization of neutral glycosphingolipids (GlcCer, LacCer, Gb3Cer, Gb4Cer, and IV3 alpha GalNAc-Gb4Cer) and monosialogangliosides [GM3(NeuAc or NeuGc), GM2 (NeuAc or NeuGc), and GM1 (NeuAc or NeuGc)]. Mixtures of the purified neutral glycolipids and monosialogangliosides were subjected to HPLC on a silica gel column, with programmed elution with isopropanol-n-hexane-water, with or without ammonium hydroxide. In order to obtain mass spectra and mass chromatograms of individual components, effluent from the HPLC column was mixed with a methanol solution of triethanolamine, which was used as the matrix for the FAB ionization, and one-thirtieth of the effluent mixture was introduced into a mass spectrometer through a frit interface. A mixture of the five neutral glycolipids, 5 micrograms of each, gave five peaks on a mass chromatogram obtained by monitoring of the corresponding major pseudo-molecular ions. A mixture of the six monosialogangliosides, 5 micrograms of each, gave six peaks on a mass chromatogram obtained by monitoring of the major pseudo-molecular ions, indicating that GM3, GM2, and GM1 were clearly separated, and that separation due to differences in sialic acid species was also achieved. In the mass spectra of the neutral glycolipids and monosialogangliosides, pseudo-molecular ions and fragment ions due to the elimination of sugar moieties were clearly detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycolipids of human large intestine: difference in glycolipid expression related to anatomical localization, epithelial/non-epithelial tissue and the ABO, Le and Se phenotypes of the donors

Human large intestine specimens were obtained during elective surgery from donors of known blood group ABO, Lewis and secretor phenotypes. The intestinal epithelial cells were isolated from the non-epithelial tissue in one case and in another case mucosa tissue was obtained by scraping. Total non-acid glycolipid and ganglioside fractions were isolated from the tissue specimens, analyzed by thin-layer chromatography and detected by chemical reagents and autoradiography after staining the plate with various blood group monoclonal antibodies and bacterial toxins. The amount of non-acid glycolipids present in the large intestine epithelial cells was 3.9 micrograms/mg of cell protein and in the non-epithelial tissue 0.39 mg/g dry tissue weight. The epithelial cells contained monoglycosylceramides and blood group Lea pentaglycosylceramides as major compounds together with small amounts of diglycosylceramides. In addition, trace amounts of tri- and tetra-glycosylceramides together with more complex glycolipids were present. The non-epithelial tissue contained mono-, di-, tri- and tetra-glycosylceramides as major non-acid components. Blood group ABH glycolipids were present in trace amounts in the non-epithelial part of the large intestine. Lea pentaglycosylceramide was the major blood group glycolipid present in all Le-positive individuals independent of the secretor status. Leb glycolipids were present in trace amounts in secretor individuals but completely lacking in non-secretors. Trace amounts of X antigens were found in all individuals, while Y antigens were only present in secretor individuals. The Lea, Leb, X and Y glycolipids were located in the epithelial cells. The gangliosides were present mainly in the non-epithelial tissue (65-350 nmol of sialic acid/g dry weight) and only trace amounts (less than 0.014 nmol/mg of cell protein) were found in the epithelial cells. The major gangliosides of the non-epithelial tissue were identified as GM3, GM1, GD3, GD1b, GT1b and GQ1b. In addition, several minor gangliosides were also present. Binding of cholera toxin to the thin-layer plate revealed trace amounts of the GM1 ganglioside in the epithelial cell ganglioside fraction.     

Syngeneic monoclonal antimelanoma antibodies and their application for analysis of tumor antigens, gene cloning, and in vitro/in vivo diagnosis

In this article, we summarized syngeneic monoclonal antimelanoma antibodies and their application for chemical characterization of mouse melanoma antigens, cloning of genomic DNA controlling antigen expression, and in vivo/in vitro tumor diagnosis. The melanoma antigen is composed of a protein complex in association with GM3(NeuAc)-like sugar moiety. The GM3 structure expresses the cross-species epitopes shared in various mammalian species, whereas the mouse specific melanoma epitope is present on protein molecules. By using the monoclonal antimelanoma reactive with GM3 epitope, we developed a very sensitive sandwich radioimmunoassay system detecting soluble melanoma antigens equivalent to 10(2)-10(3) cells/ml. The antibody was also useful in imaging tumor in vivo. These results indicate that the antibody with cross-species reactivity has a potential for tumor targeting. The monoclonal antibody M562 recognizing protein molecule with species specific epitope but not other antimelanoma antibodies, however, effectively inhibited experimental lung metastasis of melanoma cells, indicating that the M562 epitope seems to possess important biological functions. Recently, the genomic DNA controlling the antigen expression was successfully isolated by DNA transfection and expression technique with monoclonal anti-melanoma M562 and the fluorescence-activated cell sorter. We also found that genomic DNA possesses transformation-related activity in NIH3T3 cells.     

Change in the topographical distribution of GM3 during cell spreading and growth: immunostaining with monoclonal antibody against GM3

A monoclonal antibody, M2590, that recognizes hematoside (GM3) was used to analyze the immunostaining localization of GM3 of the surface of transformed and non-transformed hamster embryo fibroblasts and B16 melanoma cells. The reactivity of GM3 with the antibody changed markedly depending on the cell density. At the sparse density cells were clearly made visible by the antibody, but at the confluency the accessibility of the antibody to GM3 was greatly decreased. This density dependent change in the reactivity of GM3 was found for both normal and transformed cells. The staining pattern of GM3 was examined in relation to the actin fibers made visible with NBD-Phallacidin during cell spreading. When the cells were still round, the GM3 on microspikes or blebs was highly reactive with the antibody, and by the time cells showed circumferential staining of their actin fibers, GM3 had been distributed over the entire cell surface as punctuated spots. GM3 also was visible in substrate attachment materials (SAM). Trypsin treatment of SAM diminished the reactivity of GM3 with the antibody. The antibody did not inhibit cell attachment or spreading on a substratum coated with fibronectin or laminin.     

Uncoupling of ganglioside biosynthesis by Brefeldin A

We have studied the effect of Brefeldin A (BFA), an antiviral antibiotic, on glycosphingolipid metabolism in primary cultured cerebellar cells. Cells were labeled metabolically with [14C]galactose, or pulse-labeled with precursors of glycosphingolipid biosynthesis; i.e., [14]serine, [3H]palmitic acid or [3H]sphingosine. In all cases BFA (1 microgram/ml) strongly inhibited (75-95%) ganglioside biosynthesis beyond the stage of GM3 and GD3, that is the formation of GM1, GD1a, GT1b and GQ1b. Simultaneously an accumulation of GlcCer, LacCer, GM3 and GD3 was observed (up to 2000%). These effects could be reversed fully by removal of the BFA from the culture medium. These results indicate that the LacCer-, GM3- and GD3-synthases of murine cerebellar cells are localized together on the proximal site of the Golgi apparatus, probably in the cis-Golgi compartment. It is probable that sphingomyelin synthase and some of the other glycosyltransferases involved in ganglioside biosynthesis are localized in distinct compartments beyond the cis Golgi.