A novel ganglioside isolated from renal cell carcinoma

In renal cell carcinoma (RCC), the level of higher gangliosides is correlated with degree of metastatic potential, and cell lines derived from metastatic deposits of RCC are characterized by high expression of disialogangliosides (Saito, S., Orikasa, S., Ohyama, C., Satoh, M., and Fukushi, Y. (1991) Int. J. Cancer 49, 329-334 and Saito, S., Orikasa, S., Satoh, M., Ohyama, C., Ito, A., and Takahashi, T. (1997) Jpn. J. Cancer Res. (Gann) 88, 652-659). We now report two disialogangliosides, G1 and G2, found in the RCC cell line TOS-1. G1 from TOS-1 cells was characterized as having a novel hybrid structure between ganglio-series (region I as in Structure; same as the terminal structure of ganglioside GM2), and the lacto-series type 1 (region II). The characterization was based on reactivity with various monoclonal antibodies (mAbs) with defined epitope specificity, as well as monosaccharide and fatty acid component analysis, (1)H NMR spectroscopy, and electrospray ionization mass spectrometry of the intact compound. G1 showed strong reactivity with mAb RM2, raised originally against TOS-1 cells, and weak cross-reactivity with anti-GM2 mAb MK-1-8. The antigen is hereby termed GalNAc disialosyl Lc4Cer (IV4GalNAcIV3NeuAcIII6NeuAcLc4; abbreviated GalNAcDSLc4). G2 was identified by 1H NMR and mass spectrometry as having a structure similar to Structure but without the GalNAcbeta1-->4 substitution and showed strong reactivity with mAb FH9 reported previously to be specific for disialosyl lacto-series type 1 (disialosyl Lc(4)) having vicinal alpha2-->3 and alpha2-->6 sialosyl residues, an antigen associated with human colonic cancer. Clinicopathological studies indicate that expression of these disialoganglioside antigens in RCC tissue is correlated with the metastatic potential of RCC.     

Selective terminal alpha 2-3 and alpha 2-6 sialylation of glycosphingolipids with lacto-series type 1 and 2 chains in human meconium

Human meconium was found to contain two kinds of gangliosides with the same carbohydrate sequence belonging to the lacto-series. They were detected by TLC-immunostaining with monoclonal antibodies directed to the NeuAc alpha 2-6Gal and Lc4Cer structures. One of these two gangliosides, a major one, which migrated on TLC to a position below that of standard IV3NeuAcnLc4Cer from human erythrocytes, reacted with the antibody to NeuAc alpha 2-6Gal. The other minor one, which migrated on TLC to a position corresponding to standard IV3NeuAcnLc4Cer, was detected with the antibody to Lc4Cer only when the plate, on which the individual gangliosides were separated, was subjected to prior treatment with Vibrio cholerae sialidase. The structures of the gangliosides, each identified by means of permethylation anaylsis with Vibrio cholerae sialidase. The structures of the gangliosides, each identified by means of permethylation anaylsis and enzyme treatment after isolation with antibody monitoring, were shown to be IV6NeuAcnLc4Cer for the former and IV3NeuAcLc4Cer for the latter, indicating that the lacto-series type 2 (nLc4Cer) and 1 (Lc4Cer) chains are sialylated at different linkages, alpha 2-6 and alpha 2-3, respectively. IV6NeuAcLc4Cer and IV3NeuAcnLc4Cer were not detected, even in trace amounts, on TLC-immunostaining with the monoclonal antibodies. The concentrations of IV6NeuAcnLc4Cer and IV3NeuAcLc4Cer were 448 and 18 nmol/g dry wt of human meconium.     

Novel fucolipids of human adenocarcinoma: disialosyl Lea antigen (III4FucIII6NeuAcIV3NeuAcLc4) of human colonic adenocarcinoma and the monoclonal antibody (FH7) defining this structure

A new fucoganglioside, disialosyl Lea, has been found in the disialoganglioside fraction of human colonic adenocarcinoma. The ganglioside has been isolated from four other disialogangliosides by high-performance liquid chromatography followed by preparative high-performance thin-layer chromatography. The structure of the antigen was characterized by its conversion to lactofucopentaosyl(II)ceramide (Lea-active ceramide pentasaccharide), methylation analysis, and high-mass range electron-impact as well as field-desorption mass spectrometry of the permethylated derivative, as shown below: (Formula; see text) Specific removal of alpha 2----3-linked sialosyl residue by influenza virus A2 sialidase, or preferential hydrolysis of the same residue by Clostridium perfringens sialidase in the absence of detergent, resulted in the formation of an intermediate product, monosialosyl LeaII (III4FucIII6NeuAcLc4), which reacted with anti-Lea antibody and with monoclonal antibody FH7 and may have a sialic acid linked at the 6 position of GlcNAc. The IgG3 monoclonal antibody FH7 was established, which reacts specifically with disialosyl Lea and monosialosyl LeaII as above, but does not react with disialosyllactotetraosylceramide (IV3NeuAcIII6Neu-AcLc4), monosialosyl LeaI (IV3NeuAcIII4FucLc4), and other mono- and disialogangliosides isolated from the same cancer tissue. The antibody FH7 may be useful in the detection of human cancer.     

III6NeuAcLc4Cer in human SW1116 colorectal carcinoma cells: a possible oncofetal antigen that is not dependent on Lewis gene expression

Monospecific rabbit antibodies directed against the human milk sialyloligosaccharides III6NeuAcLcOse4 (sialyltetrasaccharide b), IV3NeuAcLcOse4 (sialyltetrasaccharide a), and IV6NeuAcnLc4Ose (sialyltetrasaccharide c) were used to detect their homologous haptens as gangliosides or ganglioside-derived sialyloligosaccharides from the human colorectal carcinoma cell line SW1116. III6NeuAcLc4Cer was first detected in human meconium [P. A. Prieto and D. F. Smith (1985) Arch. Biochem. Biophys. 241, 281-289], and its presence in a total ganglioside fraction of SW1116 cells together with its absence from a total lipid extract of normal human intestinal mucosa are consistent with III6NeuAcLc4Cer being a tumor-associated oncofetal antigen. IV3NeuAcLc4Cer, a ganglioside in human meconium [P. A. Prieto and D. F. Smith (1986) Arch. Biochem. Biophys. 249, 243-253], was also detected in SW1116 cells; an observation that is consistent with its being the immediate precursor to the sialyl-Lea ganglioside in SW1116 cells. Specific antisera against sialylated type 1 oligosaccharide chains whose expression is independent of the Lewis gene fucosyltransferase may be useful diagnostic reagents for oncofetal, carbohydrate antigens.     

A novel disialoganglioside (IV3NeuAcIII6NeuAcLc4) of human adenocarcinoma and the monoclonal antibody (FH9) defining this disialosyl structure

This ganglioside is highly immunogenic, and immunization of mice with this disialoganglioside fraction coated on Salmonella minnesota followed by fusion of immunized spleen cells with mouse myeloma and selection of the hybridoma by positive reactivity with the purified disialoganglioside resulted in the establishment of a hybridoma secreting immunoglobulin G2a antibody FH9 that reacts specifically with the ganglioside antigen above but not with monosialosyllactotetraosylceramide I (IV3NeuAcLc4), monosialosyllactotetraosylceramide II (III6NeuAcLc4), or any other gangliosides tested.     

Enhanced binding of the neural siglecs, myelin-associated glycoprotein and Schwann cell myelin protein, to Chol-1 (alpha-series) gangliosides and novel sulfated Chol-1 analogs

Extended glycoconjugate binding specificities of three sialic acid-dependent immunoglobulin-like family member lectins (siglecs), myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), and sialoadhesin, were compared by measuring siglec-mediated cell adhesion to immobilized gangliosides. Synthetic gangliosides bearing the alpha-series determinant (NeuAc alpha2,6-linked to GalNAc on a gangliotetraose core) were tested, including GD1alpha (IV(3)NeuAc, III(6)NeuAc-Gg(4)OseCer), GD1alpha with modified sialic acid residues at the III(6)-position, and the "Chol-1" gangliosides GT1aalpha (IV(3)NeuAc, III(6)NeuAc, II(3)NeuAc-Gg(4)OseCer) and GQ1balpha (IV(3)NeuAc, III(6)NeuAc, II(3)(NeuAc)(2)-Gg(4)OseCer). The alpha-series gangliosides displayed enhanced potency for MAG- and SMP-mediated cell adhesion (GQ1balpha > GT1aalpha, GD1alpha > GT1b, GD1a > GM1 (nonbinding)), whereas sialoadhesin-mediated adhesion was comparable with alpha-series and non-alpha-series gangliosides. GD1alpha derivatives with modified sialic acids (7-, 8-, or 9-deoxy) or sulfate (instead of sialic acid) at the III(6)-position supported adhesion comparable with that of GD1alpha. Notably, a novel GT1aalpha analog with sulfates at two internal sites of sialylation (NeuAcalpha2,3Galbeta1,4GalNAc-6-sulfatebeta1, 4Gal3-sulfatebeta1,4Glcbeta1,1'ceramide) was the most potent siglec-binding structure tested to date (10-fold more potent than GT1aalpha in supporting MAG and SMP binding). Together with prior studies, these data indicate that MAG and SMP display an extended structural specificity with a requirement for a terminal alpha2, 3-linked NeuAc and great enhancement by nearby precisely spaced anionic charges.     

Generation of one set of monoclonal antibodies specific for b-pathway ganglio-series gangliosides.

We established six murine monoclonal antibodies (MAbs) specific for b-pathway ganglio-series gangliosides by immunizing C3H/HeN mice with these purified gangliosides adsorbed to Salmonella minnesota mutant R595. The binding specificities of these MAbs were determined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatogram. These six MAbs, designated GGB19, GMR2, GMR7, GGR12, GMR5, and GGR13 reacted strongly with the gangliosides GD3, O-Ac-GD3, GD2, GD1b, GT1b, and GQ1b, respectively, that were used as immunogens. All these MAbs except GGB19 showed highly restricted binding specificities, reacting only with the immunizing ganglioside. None of other various authentic gangliosides or neutral glycolipids were recognized. On the other hand, MAb GGB19 exhibited a broader specificity, cross-reacting weakly with O-Ac-GD3, GQ1b, and GT1a, but not with other gangliosides or neutral glycolipids. Using these MAbs, we determined the expression of these gangliosides, especially GD1b, GT1b, and GQ1b on mouse, rat, and human leukemia cells. GD1b was expressed on rat leukemia cells, but not on mouse and human leukemia cells tested. Neither GT1b nor GQ1b was detected in these cell lines.     

Novel polysialogangliosides of skate brain structural determination of tetra, penta and hexasialogangliosides with a NeuAc-GalNAc linkage.

The gangliosides in the brain of a cartilaginous fish, skate (Bathyraja smirnovi), have been isolated and characterized by means of methylation analysis, antibody binding, enzymatic hydrolysis and MALDI-TOF MS. In addition to gangliosides with known structures (GM2, fucosyl-GM1, GD3, GD2, GT3 and GT2), five polysialogangliosides were isolated and characterized as having the following structures. (1) IV3NeuAc, III6NeuAc, II3NeuAc-Gg4Cer; (2) IV3NeuAc2, III6NeuAc, II3NeuAc-Gg4Cer; (3) IV3NeuAc, III6NeuAc, II3NeuAc2-Gg4Cer; (4) IV3NeuAc, III6NeuAc, II3NeuAc3-Gg4Cer; and (5) IV3NeuAc2, III6NeuAc, II3NeuAc3-Gg4Cer. These structures are 'hybrid-type' which comprise combinations of alpha-series and either a, b or c-series structures. Three gangliosides (2), (4) and (5), were novel. The main features of the ganglioside composition of skate brain were an abundance of gangliotriaosyl species, a lack of gangliotetraosyl species (except fucosyl-GM1), and an abundance of hybrid-types. These characteristics closely resemble those in shark brain which we reported previously [Nakamura, K., Tamai, Y. & Kasama, T. (1997) Neurochem. Int. 30, 593-604]. Two of the hybrid-type gangliosides (1) and (4), were examined for their neuritogenic activity toward cultured neuronal cells (Neuro-2A), and were found to have more potent activity than nonhybrid-type gangliosides such as GM1.     

Involvement of highly sulfated chondroitin sulfate in the metastasis of the Lewis lung carcinoma cells

The altered expression of cell surface chondroitin sulfate (CS) and dermatan sulfate (DS) in cancer cells has been demonstrated to play a key role in malignant transformation and tumor metastasis. However, the functional highly sulfated structures in CS/DS chains and their involvement in the process have not been well documented. In the present study, a structural analysis of CS/DS from two mouse Lewis lung carcinoma (3LL)-derived cell lines with different metastatic potentials revealed a higher proportion of Delta(4,5)HexUA-GalNAc(4,6-O-disulfate) generated from E-units (GlcUA-GalNAc(4, 6-O-disulfate)) in highly metastatic LM66-H11 cells than in low metastatic P29 cells, although much less CS/DS is expressed by LM66-H11 than P29 cells. This key finding prompted us to study the role of CS-E-like structures in experimental lung metastasis. The metastasis of LM66-H11 cells to lungs was effectively inhibited by enzymatic removal of tumor cell surface CS or by preadministration of CS-E rich in E-units in a dose-dependent manner. In addition, immunocytochemical analysis showed that LM66-H11 rather than P29 cells expressed more strongly the CS-E epitope, which was specifically recognized by the phage display antibody GD3G7. More importantly, this antibody and a CS-E decasaccharide fraction, the minimal structure recognized by GD3G7, strongly inhibited the metastasis of LM66-H11 cells probably by modifying the proliferative and invading behavior of the metastatic tumor cells. These results suggest that the E-unit-containing epitopes are involved in the metastatic process and a potential target for the diagnosis and treatment of malignant tumors.     

Theoretical studies on the conformation of monosialogangliosides and disialogangliosides

The preferred conformation of gangliosides GM3, GM2, GM1, GD1a and GD1b have been studied by computing their potential energies. The conformation of NeuNAc in GM3 differs from that expected for the same residue in GM2 and GM1. The NeuNAc residues in GM2 and GM1 exhibit identical conformations. Theory predicts that the terminal NeuNAc of GD1a is conformationally similar to that of GM3 and that the internal one is similar in conformation to those present in GM2 and GM1 in agreement with NMR studies. The differences in chemical shifts of the C2 and C3 carbons of the internal and terminal NeuNAc of GD1a have been attributed to differences in orientation. The present studies suggest that the binding site of cholera toxin is much smaller than that of tetanus toxin. The preferred shape of these gangliosides correlate well with their biological properties.     

Monoclonal antibodies raised against NeuAc alpha 2-6neolactotetraosylceramide detect carcinoma-associated gangliosides

Monoclonal antibodies wee obtained by the immunization of mice with 6'LM1 (IV6NeuAc-nLcOse4Cer) adsorbed to Salmonella minnesota. The monoclonal antibodies showed a specificity for gangliosides with a terminal NeuAc alpha 2-6Gal substitution, which was demonstrated in solid-phase binding assay and in liposome inhibition assay. Gangliosides with a NeuAc alpha 2-6Gal substitution were minor components of different normal tissues. However, these gangliosides were enriched in carcinomas of many tissues, and were particularly enriched in most colorectal carcinomas and in lung carcinomas. 6'LM1 is a characteristic ganglioside in fetal intestinal mucosa (meconium). This ganglioside and other gangliosides with a terminal NeuAc alpha 2-6Gal substitution might represent oncofetal antigens expressed in carcinomas owing to an activation of a "fetal' sialyltransferase.     

Induction of cellular immunity by anti-idiotypic antibodies mimicking GD2 ganglioside

Gangliosides are potentially useful targets for tumor destruction by antibodies. However, the role of gangliosides in T cell-mediated immunity to tumors is unclear. We produced three murine monoclonal anti-idiotypic antibodies (Ab2) against a monoclonal antibody (Ab1) that binds strongly to melanoma-associated GD2 ganglioside and weakly to GD3 ganglioside. All three Ab2 induced anti-anti-idiotypic antibodies (Ab3) with Ab1-like binding specificity to tumor cells and antigen in rabbits. The Ab3 specifically bound to GD2(+) tumor cells and isolated GD2, and shared idiotopes with the Ab1. Two of the three Ab2 induced GD2-specific delayed-type hypersensitivity responses in BALB/c and C57BL/6 mice, but not in C57BL/6/CD4(-/-) mice. Peripheral blood mononuclear cells (PBMC) from a melanoma patient proliferated specifically in response to in vitro stimulation with Ab2. Proliferation was accompanied by Th1-type cytokine production. Our studies demonstrate the induction of ganglioside-specific T cell-dependent immunity by Ab2 in mice. These T cells showed specific reactivity to ganglioside expressed by tumor cells.     

Enhancement of metastatic potential of mouse B16-melanoma cells to lung after treatment with gangliosides of B-16-melanoma cells of higher metastatic potential to lung

Mouse B16LuF1 melanoma cells of lower metastatic potential to lung were treated in vitro with same concentration (50 microM) of gangliosides isolated from B16LuF5, B16LuF9 or B16LuF10 cells with higher metastatic potential to lung (LuF1< LuF5< LuF9< LuF10) and injected to groups of normal mice through tail vein. The number of metastatic tumor nodules formed in lung increased in mice receiving B16LuF5, B16LuF9 and B16LuF10-ganglioside-treated B16LuF1 cells compared to mice receiving B16LuF1 cells without any ganglioside treatment. Metastatic potential of B16LuF1 cells gradually increased after treatment with gangliosides of B 16-melanoma cells of increasing metastatic potential to lung. The six major gangliosides isolated from B16LuF10 cells corresponded with standard gangliosides GT1b, GD1b, GD1a, GM1, GM2 and GM3 respectively on TLC-analysis. When B16LuF1 cells were treated in vitro with each of these six individual gangliosides and injected to groups of normal mice through tail vein the number of tumor nodules formed in lung varied. The four groups of mice receiving B16LuF1 cells treated with each of four gangliosides corresponding to GT1b, GD1b, GD1a or GM1 produced lung metastasis comparable to that of untreated control group. Only remaining two gangliosides which corresponded with standard gangliosides GM2 and GM3 increased metastatic potential of B16LuF1 cells. Thus, these results indicated that gangliosides GM2 and GM3 of B16-melanoma cells are definitely associated with metastatic potential of these tumor cells.     

Structural characterization of the disialogangliosides of murine peritoneal macrophages

Sialoglycosphingolipids (gangliosides) have been increasinglyimplicated as regulators of membrane signaling events. Macrophageganglioside patterns dramatically increase in complexity whenmurine peritoneal macrophages are stimulated in vivo with theappearance of the sialidase-sensitive monosialoganglioside GMlb(cisGMl) as a major component Gangliosides from stimulated murineperitoneal macrophages were separated into monosialo and polysialofractions and the polysialo fraction structurally characterizedby enzymatic, chemical, and mass spectra methods. All detectablecomponents of the polysialo fraction were determined to be disialogangliosides.Treatment of the polysialo fraction with Clostridium perfringenssiali-dase produced mostly the sialidase-resistant monosialoganglioside,GMIa, and a minor amount of asiaJoGMI. Perio-date oxidationand mass spectrometry analyses demonstrated the lack of tandemdisialo moieties which indicated the absence of GD1b or GD1c(GDI) entities. The combined data showed the major disialogangliosidesconsisted of GDla entities comprising IV3-NeuAc,II3NeuAc-GgOse4Cer,IV3-NeuGc,II3NeuAc-GgOse4Cer, IV3NeuAc,II3NeuGc-GgOse4Cer, andIV3-NeuGc,II3NeuGc-GgOse4Cer. Minor components consisted ofGDl entities, IV3NeuAc, III6NeuAcGgOse4Cer, IV3NeuGc, III6NeuGcGgOse4Cer,and also positional iso-mer(s) of GDl(NeuAc, NeuGc). These isomericcomponents were identified by collision analysis and tandemmass spectrometry. Consistent with previous analyses, the cer-amideportion of all polysialo (disialo) gangliosides contained solelyC18 sphingosine with C16 and C24 fatty acid moieties. Theseresults, combined with the previous characterization of macrophagemonosialogangliosides, indicate normal murine macrophage gangliosidebiosynthesis proceeds along the "a" ganglioside pathway, e.g.,GM3GM2GMlaGDl, and the proposed asialogan-glioside or "" pathway,asialoGMlGMlbGDl. The presence of totally sialidase-sensitivegangliosides appears to be characteristic of functional murineperitoneal macrophages while they are reduced in geneticallyimpaired cells. ganglioside GDla ganglioside GDl murine macrophages tandem mass spectrometry collision induced dis-association electrospray ionization     

Novel fucolipids accumulating in human adenocarcinoma. III. A hybridoma antibody (FH6) defining a human cancer-associated difucoganglioside (VI3NeuAcV3III3Fuc2nLc6)

A new fucoganglioside, 6B, which accumulates in human colonic adenocarcinoma but is absent in normal colonic mucosa, was isolated from a monosialoganglioside fraction of colonic adenocarcinomas. The structure of this ganglioside was identified as shown below by methylation analysis, direct probe mass spectrometry, and enzymatic degradation followed by examination of the degradation products with specific monoclonal antibodies. (formula; see text) The hybridoma (FH6) secreting a monoclonal IgM antibody directed to this glycolipid was selected by reactivity of the antibody with this ganglioside and lack of reactivity with other glycolipids having a closely related structure, such as sialosyllactoneotetraosylceramide (IV3NeuAcnLc4), sialosyllactofucopentaosy(III)ceramide (IV3NeuAcIII3FucnLc4), sialosyllactofucopentaosy(II)ceramide (sialosyl-Lea glycolipid; IV3NeuAcIII4FucLc4), and 6C fucoganglioside (sialosyl 2 leads to 6 fucoganglioside; VI6NeuAcIII3FucnLc6). The antibody was highly reactive with a large variety of human cancer cells, but was less reactive or did not react with a variety of normal cells.     

In Vivo and In Vitro Expression of Gangliosides in Chick Retina Müller Cells

The expression of gangliosides of the lactosylceramide (LC) and of the gangliotetraosylceramide (GTC) series on the surface of cells from the chick neural retina was investigated by double-color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosyl-GTC) was detected using the cholera toxin-cholera toxin antibody experimental approach, after conversion of polysialosyl-GTC to GM1 by treatment of the cells with neuraminidase. In retinas from 6-day-old embryos (R6), most cells (approximately 80%) expressed GD3 but not GTC. After culturing for 7 days, (R6+7), the expression of GTC was found confined to neuron-like cells; flat cells derived from Müller cells expressed GD3 but were negative for GTC expression. On the other hand, postmitotic Müller cells obtained from 13-day-old embryo (R13) or 1-day-old hatched chick retina (RP1) expressed GD3, GM1, and polysialosyl-GTC but were unable to maintain the expression of these GTCs when kept in culture for several days. According to these results, retinal cells can be defined on the basis of their ganglioside expression as follows: (a) retinoblasts, by the expression of GD3; (b) postmitotic neuronal cells, by the expression of GTC; and (c) postmitotic Müller cells, by the expression of GD3 and GTC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutral glycosphingolipids and gangliosides from spleen T lymphoblasts of genetically different inbred mouse strains

The gangliosides GM1b, GalNAc-GM1b and GD1α are typical compounds of concanavalin A stimulated splenic T lymphoblasts of CBA/J inbred mice. Their structural characterization has been described in previous studies. The intention of this work was the comparative TLC immunostaining analysis of the glycosphingolipid composition of lectin stimulated splenic T lymphoblasts obtained from six genetically different inbred mouse strains. The strains examined were AKR, BALB/c, C57BL/6, CBA/J, DBA/2 and WHT/Ht, which are commonly used for biochemical and immunological studies. The neutral glycosphingolipid GgOse4Cer, the precursor for GM1b-type gangliosides, was expressed by all six strains investigated. AKR, C57BL/6 and DBA/2 showed high and BALB/c, CBA/J and WHT/Ht diminished expression in T lymphoblasts, based on single cell calculation. The gangliosides GM1b and GalNAc-GM1b, elongation products of GgOse4Cer, displayed strain-specific differences in their intensities, which were found to correlate with the intensities of GgOse4Cer expression of the same strains. Concerning sialic acid substitution of gangliosides, GM1b and GalNAc-GM1b predominantly carry N-acetylneuraminic acid, whereas choleragenoid receptors GM1a and Gal-GalNAc-GM1b, which are also expressed by all six strains, are characterized by dominance of N-glycolylneuraminic acid. Two highly polar gangliosides, designated with X and Y, which have not been previously recognized in murine lymphoid tissue, were detected by positive anti-GalNAc-GM1b antibody and choleragenoid binding, respectively. Both gangliosides were restricted to AKR, DBA/2 and C57BL/6 mice. The other three strains BALB/c, CBA/J and WHT/Ht are lacking these structures. In summary, the GM1b-type pathway is quite active in all six strains analysed in this study. Strain-specific genetic variations in T lymphoblast gangliosides were observed with the occurrence of gangliosides X and Y. This study and data from other groups strongly indicate for GM1b-type gangliosides a functional association with T cell activation and leukocyte mediated reactions. Abbreviations: ConA, concanavalin A; GSL(s), glycosphingolipid(s); HPTLC, high-performance thin-layer chromatography; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations (1977) [48] and the ganglioside nomenclature system of Svennerholm [49] for GM1a-type gangliosides. Glucosylceramide or GlcCer, Glcβ1-1Cer; lactosylceramide or LacCer, Galβ1-4Glcβ1-1Cer; gangliotriaosylceramide or GgOse3Cer or Gg3, GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliotetraosylceramide or GgOse4Cer or Gg4, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliopentaosylceramide or GgOse5Cer, GalNAcβ1-4Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliohexaosylceramide or GgOse6Cer, Galβ1-3GalNAcβ1-4Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer. GM3, II3NeuAc-LacCer; GM1 or GM1a, II3NeuAc-GgOse4Cer; GM1b, IV3NeuAc-GgOse4Cer; GalNAc-GM1b, IV3NeuAc-GgOse5Cer; GD1a, IV3NeuAc, II3NeuAc-GgOse4Cer; GD1b, II3(NeuAc)2-GgOse4Cer; GD1c, IV3(NeuAc)2-GgOse4Cer; GD1α, IV3NeuAc, III6NeuAc-GgOse4Cer. Only NeuAc-substituted gangliosides are presented in this list of abbreviations This revised version was published online in November 2006 with corrections to the Cover Date.     

Stimulation of T lymphocyte proliferation by monoclonal antibodies against GD3 ganglioside

Cell-surface gangliosides are presumed to play a role in cell growth and differentiation. With the use of monoclonal antibodies directed against GD3, a disialoganglioside expressed predominantly by cells of neuroectodermal origin, we have found that GD3 is expressed by a subpopulation of cells of the immune system including: 1) fetal thymocytes in subcortical regions and near vessels, 2) lymph node lymphocytes in interfollicular areas and near vessels, and 3) a small subset of T cells in the peripheral blood. Mouse monoclonal antibodies (two IgGs, one IgM, and F(ab')2 fragments) reacting with GD3 were found to stimulate proliferation of T cells derived from peripheral blood. Proliferation of T cells was observed even in cultures depleted of macrophages, suggesting that activation by anti-GD3 was not dependent on the presence of accessory cells. T cell proliferation was maximum between days 5 and 7 of stimulation and was preceded by expression of interleukin 2 receptors. No stimulation was observed with control antibodies of the identical isotype or with monoclonal antibodies recognizing the gangliosides GD2 or GM2. During stimulation by anti-GD3 monoclonal antibodies, there was an expansion of the GD3+ pool of T cells, but depletion of GD3+ T cells prior to stimulation abrogated the response. Proliferation induced by binding to GD3 could be augmented by exogenous interleukin 2 and phytohemagglutinin. Anti-CD3 (T3) monoclonal antibodies had little or no effect. These results demonstrate that binding to GD3 on the surface of T cells can elicit signals for T cell proliferation.     

Monoclonal antibodies to disialogangliosides: characterization of antibody-mediated cytotoxicity against human melanoma and neuroblastoma cells in vitro

We previously reported the binding specificities of two anti-ganglioside GD2 murine monoclonal antibodies (MAbs), A1-425 and A1-267, both of which are of IgG3 isotype. A1-425 reacts specifically with ganglioside GD2, whereas A1-267 binds preferentially to GD2 but also reacts with GD3 [Tai, T., Kawashima, I., Tada, N., & Dairiki, K. (1988) J. Biochem. 103, 682-687]. In this paper, they were used for comparative analyses of antibody-mediated cytotoxicity, i.e., antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human melanoma and neuroblastoma cell lines. Melanoma cells were found to contain GD2 and/or GD3, whereas neuroblastoma cells expressed only GD2. Both antibodies induced high levels of ADCC and CDC to GD2/GD3-positive cells with human peripheral large granular lymphocytes (LGL) as effector cells and in the presence of human serum, respectively. A good correlation was obtained between the contents of disialogangliosides and the binding level of the antibodies; both melanoma and neuroblastoma cells with larger amounts of GD2/GD3 showed a higher level of antibody binding than did the cells with a smaller amount of GD2/GD3. Surprisingly, ADCC did not correlate well with the binding level of the antibodies. Thus, A1-425 showed stronger lytic activity than A1-267 in spite of the binding level of A1-425 being similar to or lower than that of A1-267 on the cell surfaces. Antigen-antibody complexes composed of GD2 and A1-425 showed higher binding levels to LGL than complexes of GD2 and A1-267. In contrast, free MAb molecules gave minimum binding to LGL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gangliosides enhance migration of mouse B16-melanoma cells through artificial basement membrane alone or in presence of laminin or fibronectin

The migration of B16LuF1 cells, B16-melanoma cells of lower metastatic potential to lung was enhanced through artificial basement membrane in presence of gangliosides of B16LuF1 cells as well as gangliosides of B16-melanoma cells of higher metastatic potential to lung, namely, B16LuF5 and B16LuF10 cells. The same concentration (50 microM) of gangliosides of B16LuF1, B16LuF5 and B16LuF10 cells gradually increased the migration of B16LuF1 cells through basement membrane. Moreover, B16LuF10 cell gangliosides modified the migratory effect of laminin and fibronectin on B16LuF1 cells. Laminin alone increased migration of B16LuF1 cells whereas fibronectin alone decreased migration of the same cells. When B16LuF10 cell gangliosides were used in combination with fibronectin, gangliosides removed the migration inhibitory effect of fibronectin resulting in net enhancing effect. Gangliosides in association with laminin also increased the enhancing effect of laminin on migration of B16LuF1 cells. Thus, gangliosides showed additive enhancing effect when used in combination with laminin. However, effect of individual gangliosides were different. Out of six gangliosides isolated from B16LuF10 cells only two gangliosides corresponding to standard gangliosides GM2 and GM3 enhanced migration of B16LuF1 cells. The migration of B16LuF1 cells in presence of each of the remaining four gangliosides corresponding to GT1b, GD1b, GD1a and GM1 was not altered and was comparable to that of untreated control. Thus, gangliosides of B16 melanoma cells alone or in combination with laminin or fibronectin enhanced migration of B16 melanoma cells through artificial basement membrane, suggesting possible role of tumor gangliosides during invasion of metastatic tumor cells through basement membrane of the surrounding tissues in vivo.