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.     

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.     

Acidic glycosphingolipids of human amnion

Six major acidic glycosphingolipids were isolated from human amnion using DEAE Sephadex A-25 and silica beads column chromatography. The structures of these glycosphingolipids were determined by methylation analysis, TLC immunostaining and/or negative ion FAB-MS, and were concluded to be II3 alpha NeuAcLacCer(GM3), IV3 alpha NeuAcnLc4-Cer (sialyl[alpha 2-3]paragloboside), IV6 alpha NeuAcnLc4Cer (sialyl[alpha 2-6]paragloboside), IV3 alpha NeuAcIII4 alpha FucLc4Cer (sialyl Lea), VI3 alpha NeuAcnLc6Cer (i-ganglioside) and II3 alpha (NeuAc alpha 2----8NeuAc)LacCer (GD3). In addition, several minor glycosphingolipids were detected with specific monoclonal antibodies, including glycolipids with NeuAc alpha 2-3Gal beta 1-4GlcNAc-beta 1- or NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1- determinant. Our results show that the glycosphingolipids of human amnion are characterized by having mainly type II chain analogues and onco-fetal antigens.     

Extended type 1 chain glycosphingolipids: dimeric Lea (III4V4Fuc2Lc6) as human tumor-associated antigen

Glycolipid extracts from various human cancer tissues and cell lines showed the presence of a slow-migrating glycolipid component which was strongly reactive with monoclonal antibody (mAb) NCC-ST-421 (raised against human gastric adenocarcinoma) and weakly cross-reactive with anti-Lea mAbs. The slow-migrating glycolipid was isolated from human colonic adenocarcinoma cell line Colo205 grown in nude mice, and was purified by high-performance liquid chromatography followed by preparative thin-layer chromatography. Its structure was elucidated by sequential enzymatic degradation and thin-layer chromatography immunostaining of the degradation products with various mAbs, 1H NMR spectroscopy, positive-ion fast atom bombardment mass spectrometry, and methylation analysis. The major slow-migrating component reacting with mAb ST-421 was identified as dimeric Lea, with the structure as follows. [formula: see text] Antigens containing this structure and various analogous structures (including enzymatically synthesized Lea/Lex hybrid antigen) were tested with ST-421. While the mAb was equally reactive with dimeric Lea and Lea/Lex, only the former was chemically detectable as the slow-migrating glycolipid from the tumor extract. ST-421 showed less reactivity with simple Lea (III4FucLc4) or extended Lea (V4FucLc6, and/or IV3Gal beta 1----3[Fuc alpha 1----4]GlcNAcnLc4), and was not reactive with Lex/Lex (dimeric Lex). It was concluded, therefore, that the major tumor-associated slow-migrating glycolipid reacting with ST-421 has the dimeric Lea structure shown above. Since extension of lacto-series structure has been shown to be limited to type 2 chain in normal cells and tissues, extended elongation of type 1 chain as shown in this structure represents a novel tumor-associated epitope.     

A sialidase-susceptible ganglioside, IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer, is a major disialoganglioside in WHT/Ht mouse thymoma and thymocytes.

The disialogangliosides of WHT/Ht mouse thymomas, which were obtained by subcutaneous transplantation of a thymoma that developed spontaneously in a WHT/Ht mouse, were purified and characterized. From the results of sugar-composition analysis, a permethylation study, enzymatic hydrolysis followed by TLC-immunostaining, negative-ion fast atom bombardment mass spectrometry (FAB/MS), and 1H-NMR spectroscopy, the structure of one of the five purified disialogangliosides was determined to be IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer. The other 4 disialogangliosides were tentatively characterized on the basis of sialidase treatment followed by TLC-immunostaining with cholera toxin B subunit and anti-Gg4Cer antibody to be IV alpha(NeuAc alpha-NeuGc)-Gg4Cer, IV alpha(NeuGc alpha-NeuAc)-Gg4Cer, IV alpha NeuAc,II3 alpha NeuAc-Gg4Cer, and IV alpha NeuGc,II3 alpha NeuGc-Gg4Cer. In addition, another component exhibiting one spot on TLC was a mixture of IV alpha NeuGc,II3 alpha NeuAc-Gg4Cer and IV alpha NeuAc,II3 alpha NeuGc-Gg4Cer. Then the occurrence of these gangliosides in WHT/Ht mouse thymocytes was examined. As one of two major disialogangliosides, the thymocytes contained IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer, which was characterized with a mass spectrum and mass chromatograms obtained by micro high-performance liquid chromatography-FAB/MS. The other major disialoganglioside was tentatively characterized to be II3 alpha-(NeuGc alpha-NeuGc)-Gg4Cer by sialidase treatment followed by TLC-immunostaining. A sialidase-susceptible monosialoganglioside, IV3 alpha NeuGc-Gg4Cer [GM1b(NeuGc)], had been reported to be characteristic of mouse immune tissues [Nakamura, K. et al. (1988) J. Biochem, 103, 201-208]. Taken together, the results suggest that the pathway from Gg4Cer to IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer through GM1b(NeuGc) is quite active in mouse immune tissues.     

Monoclonal antibody (5F3) defining renal cell carcinoma-associated antigen disialosyl globopentaosylceramide (V3NeuAcIV6NeuAcGb5), and distribution pattern of the antigen in tumor and normal tissues

Renal cell carcinoma (RCC) has been characterized by high expression of three types of disialogangliosides: two based on lacto-series type 1 structure (disialosyl Lc₄, GalNAc disialosyl Lc₄), the other based on globo-series structure (disialosyl globopentaosylceramide; disialosyl Gb5). The present study established a mAb, 5F3, directed to disialosyl Gb5. 5F3 was established after immunization with RCC cell line ACHN. The major disialoganglioside antigen isolated from ACHN cells, showing specific reactivity with 5F3, was characterized unequivocally as disialosyl Gb5 (V³NeuAcIV⁶NeuAcGb5) by identification of the core structure as globopentaosylceramide (Gb5) after enzymatic and acid hydrolysis, and by 2-dimensional ¹H-NMR spectroscopy. 5F3 does not react with monosialosyl Gb5 (V³NeuAcGb5), Gb5, or any lacto-series structures. 5F3 strongly stained 19 of 41 cases of primary RCC tissue. It reacted with proximal tubules (but not distal tubules) of kidney, microglial cells of cerebrum and cerebellum, goblet cells of stomach and intestine, smooth muscle of various organs. It did not react with parenchymatous cells of various organs, except for kidney epithelia and prostate stroma. Immunostaining of RCC tissue by mAb 5F3, in combination with staining by other antibodies directed to globo-series and lacto-series structures, has prognostic significance in defining metastatic potential of RCC.     

Novel fucolipids accumulating in human adenocarcinoma. II. Selective isolation of hybridoma antibodies that differentially recognize mono-, di-, and trifucosylated type 2 chain

A series of glycolipids having the X determinant (Gal beta 1----4 [Fuc alpha----3]GlcNAc) at the terminus and a fucosyl alpha 1----3 residue at the internal GlcNAc residue have been isolated and characterized from tumor tissues (Hakomori, S., Nudelman, E., Levery, S.B., and Kannagi, R. (1984) J. Biol. Chem. 259, 4672-4680. A series of monoclonal antibodies that differentially recognize glycolipids with mono-, di-, and trifucosylated type 2 chain have been isolated and characterized. The antibody FH4 shows a remarkable preferential reactivity towards di-/or trifucosylated type 2 chain, i.e. it does not react with monofucosylated structures, including lactofucopentaosyl (III) ceramide (III3FucnLc4), monofucosyl neolactonorhexaosylceramide (y2, V3FucnLc6), and monofucosyl neolactonoroctaosylceramide (Z1, VII3FucnLc8), but reacts well with di- and trifucosylated type 2 chain structures such as difucosyl neolactonorhexaosylceramide (III3V3Fuc2nLc6) and trifucosyl neolactonoroctaosylceramide (III3V3VII3Fuc3nLc8). Two other monoclonal antibodies, FH5 and ACFH18, preferentially react with trifucosylated type 2 chain structure (III3V3VII3Fuc3nLc8), although cross-reactivity with difucosylated type 2 chain (III3V3Fuc2nLc6) was observed. They showed a minimal cross-reaction with monofucosylated type 2 chain. In contrast, the antibody FH1 does not react with III3FucnLc4 but reacts with V3FucnLc6, III3V3Fuc2nLc6, and III3V3VII3Fuc3nLc8. Two monoclonal antibodies, FH2 and FH3, do not discriminate among various glycolipids having fucosylated type 2 chain, and their reactivities are essentially similar to previously established antibodies directed to the X determinant, such as anti-SSEA-1, WGHS 29, VEP8 and 9, My-1, etc. This series of antibodies will be useful to detect the specific type of glycolipid with fucosylated type 2 chain accumulating in human cancer and in undifferentiated cells.     

The monoclonal antibody directed to difucosylated type 2 chain (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc; Y Determinant)

One of the monoclonal (AH-6) antibodies prepared by hybridoma technique against human gastric cancer cell line MKN74 was found to react with a series of glycolipids having the Y determinant (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc). The structure of one such glycolipid isolated from human colonic cancer and from dog intestine was identified as lactodifucohexaosyl-ceramide (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide; IV3,III3Fuc2nLc4Cer). The hapten glycolipid did not react with monoclonal antibodies directed to Lea, Leb, and X-hapten structures, and the AH-6 antibody did not react with the X-hapten ceramide pentasaccharide (Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), H1 glycolipid (Fuc alpha 1 leads to 2Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), nor with glycolipids having the Leb (Fuc alpha 1 leads to 2Gal beta 1 leads to 3[Fuc alpha 1 leads 4]GlcNAc beta 1 leads to R) determinant. The antibody reacted with blood group O erythrocytes, but not with A erythrocytes. Immunostaining of thin layer chromatography with the monoclonal antibody AH-6 indicated that a series of glycolipids with the Y determinant is present in tumors and in O erythrocytes.     

Binding specificity of siglec7 to disialogangliosides of renal cell carcinoma: possible role of disialogangliosides in tumor progression

Previous studies indicate that expression of higher gangliosides in renal cell carcinoma (RCC) is correlated with metastatic potential, particularly in the lung. Out of five major gangliosides in RCC, three disialogangliosides (disialogalactosylgloboside, IV(3)NeuAcIII(6)NeuAcLc(4), and IV(4)GalNAcIV(3)NeuAcIII(6)NeuAcLc(4)) bind strongly to siglec7, which is expressed highly in monocytes and natural killer cells. Out of other gangliosides tested, 2-->6 sialylparagloboside, GD3, GD2, and GT1b, but not other lacto- or ganglio-series gangliosides, showed clear binding to siglec7. In view of preferential metastasis of RCC to the lung, and binding of RCC cell line TOS-1 to lung tissue sections as shown in our previous study, we examined expression of siglec7 in the lung. siglec7 is expressed highly in resident blood cells, but not in parenchymatous cells. TOS-1 cells aggregate together strongly through adhesion with peripheral blood mononuclear cells to form large clumps. This suggests the possibility that such aggregates may form embolisms of microvasculature, particularly in the lung, which initiate metastasis. Other possible roles of higher gangliosides in RCC in promoting metastasis and tumor progression are discussed.     

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.     

Monoclonal antibody AA4, which inhibits binding of IgE to high affinity receptors on rat basophilic leukemia cells, binds to novel alpha-galactosyl derivatives of ganglioside GD1b

Mouse monoclonal antibody AA4 inhibits the binding of IgE to high affinity IgE receptors on the rat basophilic leukemia cell line RBL-2H3. As shown by immunostaining of thin layer chromatograms, antibody AA4 binds avidly to two disialogangliosides (antigen I and antigen II) that occur in this cell line. The two antigens were purified by anion exchange chromatography followed by short-bed continuous thin-layer chromatography. About 230 micrograms of antigen I and 60 micrograms of antigen II were obtained from 20 g (wet weight) of leukemia cells. The structures of both purified antigens were determined to be alpha-galactosyl derivatives of the ganglioside GD1b by fast atom bombardment-mass spectrometry, by chemical ionization-mass spectrometry of permethylated samples, by gas chromatography-mass spectrometry of partially methylated alditol acetates, and by treatment with exoglycosidases and mild acid hydrolysis. The structure of antigen I is: (formula; see text) Antigen II has an additional alpha-galactosyl residue as follows: (formula; see text) The ceramide of antigen I contains approximately equal amounts of C24:0, C22:0, C20:0, C18:0, and C16:0 N-acyl fatty acids. The ceramide base is predominantly sphingosine along with a small amount of dihydrosphingosine. In contrast, the ceramide of antigen II contains mainly C24:0 N-acyl fatty acid with much lower amounts of C22:0, C20:0, and C18:0 fatty acids. Moreover, the ceramide base is approximately 55% sphingosine and 45% dihydrosphingosine. No unsaturated N-acyl fatty acids were detected in either antigen.     

Blood group A determinants with mono- and difucosyl type 1 chain in human erythrocyte membranes

Application of a monoclonal antibody defining monofucosyl type 1 chain A (AH21) revealed the presence of a glycolipid having the same thin-layer chromatography mobility as Aa but showing a clear reactivity with AH21. This glycolipid was detectable in Lea-b- erythrocytes but not in Lea+b- or Lea-b+ erythrocytes. Another monoclonal antibody defining difucosyl type 1 chain A (HH3) detected the presence of a glycolipid component reacting with this antibody in Lea-b+ erythrocytes but not in Lea+b- or Lea-b- erythrocytes. The component defined by monoclonal antibody AH21 and that defined by HH3 were isolated and characterized by 1H NMR spectrometry and methylation analysis as having the structures (Formula: see text) The 1H NMR spectra of these glycolipid antigens were characterized by resonances for anomeric protons that are identical with those of glycolipids with type 1 chain previously isolated but distinctively different from those of type 2 chain analogues. Resonances reflecting ceramide composition are characteristic for these antigens from human erythrocytes and are distinguishable from those of the same antigen from other sources.     

Developmental patterns of ganglioside sialosylation coincident with neuritogenesis in cultured embryonic chick brain neurons.

Chick brain precursor neurons were observed to introduce sialic acid biosynthetically into only three specific gangliosides: monosialosyl lactosyl ceramide (GM3), disialosyl lactosyl ceramide (GD3), and disialosyl gangliotrihexosyl ceramide (GD2), when sialic acid was labeled metabolically by its obligate precursor, [3H] ManNAc. Sialosyl donor CMP-[3H]NeuAc supplied in the culture medium gave rise uniquely to surface-labeled GD3. Thus sialosyl transferase/GD3 synthase activity is expressed both intraneuronally and in the neuronal exofacial surface. Upon epidermal growth factor-induced onset of neurite outgrowth, labeled complex sialosyl gangliotetrahexosyl ceramide species of gangliosides began to appear in the embryonic neuronal plasma membrane. However, intraneuronal and exofacial sialosyl transferase/GD3 synthase activities remained constant, with or without neurite outgrowth. Moreover, simpler species of gangliosides maintained a steady quantitative sialosyl level (1.6 +/- 0.2 micrograms of sialic acid/mg of protein), whereas more complex species completely absent before neurite outgrowth accrued and reached 4.8 +/- 0.9 micrograms of sialic acid/mg of protein with full neurite development. This analysis of developmental patterns of ganglioside sialosylation has provided evidence that stable neurite outgrowth depends upon generation by the neuron of special plasma membrane with a massive content of complex higher species of gangliosides.     

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.     

Lea-active heptaglycosylceramide, a hybrid of type 1 and type 2 chain, and the pattern of glycolipids with Lea, Leb, X (Lex), and Y (Ley) determinants in human blood cell membranes (ghosts). Evidence that type 2 chain can elongate repetitively but type 1 chain cannot

Two major glycolipids reactive with the monoclonal anti-Lea antibody have been isolated from human blood cell membranes. One component was identified as lactofucopentaosyl(II)ceramide and the other as a ceramide heptassaccharide with the structure described below: (formula; see text) The structure includes the Lea determinant (type 1 chain) linked to lactoneotetraosylceramide (type 2 chain); thus, it is regarded to be a hybrid between type 1 and 2 chain. In addition, a minor component having the thin-layer chromatographic mobility of a ceramide nonasaccharide, which was reactive to anti-Lea antibody, was detected. No other component with a thin-layer chromatographic mobility slower than the above components and reactive to the anti-Lea antibody was detected. In contrast, a series of slowly migrating glycolipids having X (Lex) determinant (Gal beta 1----4(Fuc alpha 1----3)GlcNAc) was detected. A similar series of long chain glycolipids having Y (Ley) determinant (Fuc alpha 1----2Gal beta 1----4(Fuc1----3)GlcNAc) was detected in human blood cells; in contrast, only one major Leb glycolipid was found with the mobility of a ceramide hexasaccharide. No glycolipid with a long carbohydrate chain composed exclusively of type 1 chain was detected. Thus, chain elongation may proceed through type 2 chain, but not through type 1 chain. Lea and X (Lex) haptens are distributed equally among blood group A, B, and O red blood cells, whereas the quantity of Leb and Y (Ley) haptens is much lower in A and B blood cells than in O blood cells.     

Immunochemistry of the Lewis blood-group system: isolation and structures of Lewis-c active and related glycosphingolipids from the plasma of blood-group O Le(a-b-) nonsecretors

Five different glycosphingolipid fractions (GL-3, 285 micrograms; GL-5, 1090 micrograms; GL-6, 615 micrograms; GL-7, 555 micrograms; and GL-8, 155 micrograms) have been isolated from 25 liters of plasma of O Le(a-b-) nonsecretors by means of ethanol extraction, several steps of Folch distribution, and reversed-phase, silicic acid, and ion-exchange column chromatography of native or peracetylated substances. Final purification, accomplished by preparative silica gel high-performance thin-layer chromatography, led to chromatographic homogeneity of GL-3 and GL-6. In the hemagglutination inhibition as well as quantitative passive hemagglutination techniques two of these substances (GL-3, GL-5) exhibited distinct, and the other three (GL-6-GL-8) very strong, Lec blood-group activities when tested against two different Lec antisera of human or goat origin. The fragments' structures were elucidated by fast atom bombardment and electron impact mass spectrometry of permethylated derivatives in order to determine molecular weight, sugar sequence, position of branching points, and type of oligosaccharide chains, as well as fatty acid and sphingosine patterns of the ceramide residue. Combined gas-liquid chromatography and mass spectrometry of partially methylated alditol acetates identified sugar composition and glycosidic linkages. Thus, the following structures could be established: (formula; see text) In contrast to the structurally homogeneous GL-3, minor amounts of 4-O-substituted GlcNAc pointed to a small contamination of GL-6 by branched type 2 ceramide nonasaccharide analogs. Glycolipids containing hepta- or nonasaccharides as in GL-3 or GL-6 could also be identified in fractions GL-5 (ceramide heptasaccharide) and GL-7 and GL-8 (ceramide nonasaccharide). These latter fractions revealed, however, distinct heterogeneity due to the presence of a small amount of either a type 2 analog of GL-3 (GL-5) or linear, mainly type 2, ceramide hexa- (GL-5, GL-7) or octasaccharides (GL-8). In addition to previous immunochemical communications the presented Lec active structures of GL-3 and GL-6 provide evidence that 3-fucosyl-N-acetyllactosamine in combination with a type 1 based oligosaccharide sequence and a 3,6-galactosyl branching point are essential parts of the Lec antigenic determinant (as marked in the formula of GL-6).     

Isolation and characterization of ganglioside GM1b from normal human brain

A sialidase-susceptible monosialoganglioside was isolated from normal human brain by DEAE-Sephadex A-25 and Iatrobeads column chromatography. The yield of this ganglioside was about 6 mg per whole brain. Its structure was elucidated by sugar analysis, sialidase digestion, permethylation, and proton NMR studies. This ganglioside had carbohydrate, fatty acid, and long-chain base compositions identical to those of brain GM1a. However, the sialosyl residue was found to be linked (alpha 2-3) to the terminal galactosyl residue of the asialo-GM1a backbone. The complete structure of this ganglioside was therefore identified as GM1b or IV3NeuAcGgOse4Cer.     

Monoclonal antibody-defined antigen of human uterine endometrial carcinomas is Leb

A monoclonal antibody, MSN-1, generated by immunizing a mouse with a human uterine endometrial adenocarcinoma cell line, SNG-II, was strongly and specifically reactive with neutral glycosphingolipids from cancer tissues of patients with uterine endometrial adenocarcinomas. The glycosphingolipid antigen was purified from pooled human meconia, which contained the antigen at the concentration of 1.95 mumol/g dry weight. Its structure was determined by NMR, negative ion FABMS, permethylation analysis, and TLC-immunostaining with monoclonal anti-Lc4Cer antibody, and was concluded to be the III4IV2Fuc2Lc4Cer,Leb antigen of the human Lewis blood system. On ELISA, the monoclonal antibody was found to be strongly reactive with Leb, slightly with Lea and not at all with A, B, H, or IV2FucGg4Cer. The amount of Leb in cancerous regions in the patients with the Lea-b+ blood group was significantly increased compared to that in normal regions in the same patients, and it was a newly appearing antigen in the cancerous tissue of a patient with the Lea+b- blood group.     

Molecular characterization and immunohistochemical localization of IV(4)GalNAcGgOse(4)Cer: a naturally occurring novel neutral glycosphingolipid in bovine brain

A pair of novel neutral glycosphingolipids (Ngsls) has been identified in bovine brain. Their mobilities on thin layer chromatography were slightly different from a standard pentaglycosylceramide (nLcOse(5)Cer from bovine erythrocytes). The compounds were purified to homogeneity by column chromatography. Their fatty acid and base compositions, their monosaccharide compositions and sugar linkage positions were determined by gas-liquid chromato-graphy/mass spectrometry. Carbohydrate sequence analy-sis by(1)H NMR spectroscopy and stepwise exoglyco-sidase digestion indicated the following pentaglycosyl structure for the oligosaccharide moiety of both Ngsls: GalNAcbeta1-4Galbeta1-3GalNAcbeta1-4Galbeta1-4Gl c. The two Ngsls (abbreviated as IV(4)GalNAcGgOse(4)Cer or GalNAc-GA1), differ in their ceramide compositions, having d18:0 and d18:1 sphingosine as their long chain bases. A monospecific polyclonal anti-GalNAc-GA1 antibody, prepared in rabbit and purified by affinity chromatography, stained the neurons of cerebral cortex and cerebellum including Purkinje cells in adult rat brain, indicating that the novel GalNAc-GA1 is associated with cerebellar and other neurons in vertebrate central nervous system.