Glycosphingolipid carriers of carbohydrate antigens of human myeloid cells recognized by monoclonal antibodies

Six monoclonal antibodies with known specificities for the carbohydrate antigens i, X or Y, and seven anti-myeloid antibodies (determinants unknown) selected for their differing reaction patterns with human leucocytes were tested in chromatogram binding assays for reactions with myeloid cell glycolipids derived from normal human granulocytes and chronic myelogenous leukemia cells. Antigenicities were found exclusively on minor glycolipids which were barely or not at all detectable with orcinol-sulphuric acid stain. Among these, a neutral glycosphingolipid bound the anti-i antibody Den and chromatographed as the ceramide octasaccharide, Gal beta 1----4GlcNac beta 1----3Gal beta 1----4GlcNac beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer. Several species of neutral glycosphingolipids with six to more than ten monosaccharides were detected which carry the X antigen and others the Y antigen: Gal beta 1----4(Fuc alpha 1----3)GlcNAc and Fuc alpha 1----2Gal beta 1----4(Fuc alpha 1----3)GlcNAc, respectively. In addition, three new types of carbohydrate specificities were detected among the myeloid cell glycolipids. Two were associated with neutral glycolipids: the first, recognised by anti-myeloid antibodies VIM-1 and VIM-10, was expressed on a distinct set of glycolipids with six or more monosaccharides, and the second, recognized by VIM-8, was expressed on glycolipids with more than ten monosaccharides. The third specificity, recognised by the anti-myeloid antibody VIM-2, was expressed on slow migrating sialoglycolipids with backbone structures of the poly-N-acetyllactosamine type that are susceptible to degradation with endo-beta-galactosidase. Thus, we conclude that the i and Y antigens occur among the glycolipids of normal myeloid and chronic myelogenous leukemia cells and that a high proportion of hybridoma antibodies raised against differentiation antigens of myeloid cells are directed at carbohydrate structures.     

Isolation and fine-structure characterization of four monoclonal antibodies reactive with glycoconjugates containing terminal GlcNAc residues: application to aspects of lacto-series tumor antigen biosynthesis.

A series of murine monoclonal antibodies, each reactive with terminal GlcNAc residues expressed on glycolipids, have been isolated after immunization with the glycolipid nLc5 (GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1---- 4Glc beta 1----1Cer). The derived antibodies, designated TE-4, TE-5, TE-6, and TE-7, were tested for binding specificity with a variety of terminal GlcNAc-containing oligosaccharides expressed on glycolipids and glycoproteins. Antibody TE-4 was found to be reactive only with linear and branched terminal GlcNAc beta 1----3Gal containing structures present in lacto-series carbohydrates irrespective of core chain length. The binding specificity of TE-7 was similar except that no reactivity was observed with the short chain structure Lc3 and was weakly reactive with branched agalacto-I structures, suggesting a longer recognition epitope than for the TE-4 antibody. Antibodies TE-5 and TE-6 reacted with terminal GlcNAc beta 1----3Gal structures and as well GlcNAc beta 1----2(6)Man structures present on BSA-oligosaccharide conjugates. Weak binding was also observed with GlcNAc beta 1----6Gal structures with these antibodies. TE-5 was found to be particularly sensitive to low amounts of terminal GlcNAc-containing glycolipids in both solid phase assays and in TLC-immunostaining studies of neutral glycolipids extracted from colonic adenocarcinoma cell lines and tumors. No reactivity was observed with internal GlcNAc residues with any antibody tested. The panel of antibodies was applied to studies of binding to Triton X-100-solubilized fractions from normal mucosal and adenocarcinoma cell lines after desialylation and Smith degradation to expose terminal GlcNAc residues on glycoproteins and glycolipids. Binding of antibodies TE-4 and TE-7 was restricted to adenocarcinoma-derived cell fractions. Application of these antibodies in studies of lacto-series core chain synthesis and in immunodiagnostic procedures after initial treatments to concentrate lacto-series antigens into terminal GlcNAc-containing structures is discussed.     

GalNAc beta 1----3 terminated glycosphingolipids of human erythrocytes

Nonacid glycosphingolipids with 4 to 10 sugar residues isolated from pooled erythrocytes of blood group O donors have been efficiently separated as peracetylated derivatives on silicic acid. This procedure enabled a quantitative estimate of individual compounds and also revealed several GalNAc beta 1----3 terminated structures. The structural characterization of these glycolipids with 1H-NMR spectroscopy, direct inlet mass spectrometry, gas chromatography, and gas chromatography-mass spectrometry identified the compounds as GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1-N-acetyl sphingosine and GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1-N-acetyl phytosphingosine, GalNAc beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1 ceramide, and GalNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1 ceramide.     

Y and blood group B type 2 glycolipid antigens accumulate in a human gastric carcinoma cell line as detected by monoclonal antibody. Isolation and characterization by mass spectrometry and NMR spectroscopy

A monoclonal antibody (mAb), BR55-2, was generated from mice immunized with MCF-7 human breast carcinoma cells. This mAb specifically detected glycolipids with the Y determinant Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)-beta 1----3Gal beta 1----4Glc beta 1----1 Cer and the Y-related B-active difucosylated determinant Gal alpha 1----3Gal(2----1 alpha Fuc) beta 1----4GlcNAc(3----1 alpha Fuc) beta 1----3Gal beta 1----4Glc beta 1----1 Cer, but was not reactive with related monofucosylated glycolipids of type 2 chain (X-antigen, blood group H), type 1 chain (Lea antigen, blood group H and B) or with difucosylated type 2 and type 1 chain structures (A blood group antigen or blood group B and Leb, respectively). A series of glycolipids with Y and blood group B type 2 determinants were detected in human gastric adenocarcinoma cell line KATO III with mAb BR55-2 and with a previously characterized anti-blood group B mAb PA83-52 (Hansson, G. C., Karlsson, K.-A., Larson, G., McKibbin, J. M., Blaszczyk, M., Herlyn, M., Steplewski, Z., and Koprowski, H. (1983) J. Biol. Chem. 258, 4091-4097). The isolated antigens were structurally characterized by mass spectrometry of permethylated and permethylated-reduced derivatives and by proton NMR spectroscopy. In a chromatogram binding assay, mAb BR55-2 and mAb PA83-52 detected minor components with slower mobility than the Y-6 and blood group B-7-type 2 structures. The detection of a B type 2 determinant is the first chemical evidence for the presence of an autologous difucosyl blood group B type 2 antigen in human adenocarcinoma cells.     

Glycolipid antigens of human polymorphonuclear neutrophils and the inducible HL-60 myeloid leukemia line

Glycolipid and cell surface carbohydrate antigens of human polymorphonuclear neutrophils (PMN) and of HL-60 myeloid leukemia cells were analyzed with a panel of defined, monoclonal anti-carbohydrate antibodies. Antigenicities of intact PMN, HL-60, and retinoic acid-induced HL-60 (r.a.-HL-60) were studied by flow cytofluorometry. These three cell populations displayed quantitative differences, some of which were induction dependent, in their expression of lactosyl, N-acetyllactosaminyl, Y-hapten (Fuc alpha 1----2Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----R), and sialosyl-X-hapten (SA alpha 2----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----R) specificities. Structures reactive with antibodies specific for long-chain mono-, and di- or tri- alpha 1----3 fucosylated lacto-series glycolipids were also detected. Glycosphingolipids purified from organic extracts of these cells were analyzed to seek information concerning the chemical basis for these surface antigenic differences, to assess the structural and antigenic diversity of PMN and HL-60 glycolipids, and to quantitate chemically and antigenically prominent glycolipids. Binding of monoclonal antibodies to thin-layer chromatograms demonstrated that each of the specificities on intact cells was carried by one or more distinct glycolipids. The abundance of immunoreactive glycolipids in the extracts paralleled the relative staining intensities of the intact cell populations. Several "cryptic" glycolipid antigens, including alpha 2----6 sialosylated structures enriched five- to 10-fold in PMN extracts, were not detected on intact cells. Lactosylceramide accounted for two-thirds of the approximately 1.5 X 10(9) glycolipid molecules contained in each PMN. The remaining glycolipid antigens appeared to include structurally diverse fucolipids, fucogangliosides, and neutral and sialosylated glycolipids with Gal beta 1----4GlcNAc beta 1----R terminal core structure. The abundance, diversity, and induction-dependent expression of these structures suggest that they may participate in PMN maturation and function.     

Biosynthetic pathways for the Leb and Y glycolipids in the gastric carcinoma cell line KATO III as analyzed by a novel assay

The biosynthetic pathways for the difucosylated type 1 and 2 glycolipids, Leb and Y, respectively, were investigated in the gastric carcinoma cell line KATO III, using a novel chromatogram binding assay. The type of fucosylation obtained was deduced from the binding pattern of monoclonal antibodies specific for the biosynthesized glycolipid products using microsomal fractions as the source of enzyme, pure glycolipids and non-radioactive GDP-fucose as acceptor and donor substrates, respectively. The Leb glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer) was synthesized mainly via the blood group H, type 1, precursor (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3LacCer). However, the Lea glycolipid (Gal beta 1----3GlcNAc(4----1 alpha Fuc)beta 1----3LacCer) also served as a precursor for the alpha 1----2 fucosyltransferase, thus allowing conversion of Lea to Leb. This biosynthetic route represents either an "aberrant" specificity of the Fuc alpha 1----2 transferase associated with these gastric carcinoma cells and/or a new member of the alpha 1----2 fucosyltransferase family. The Y glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) was synthesized exclusively via the classical pathway using the blood group H type 2 glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc beta 1----3LacCer) as precursor. The X glycolipid (Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) did not serve as an acceptor substrate for the alpha 1----2 fucosyltransferase(s) present. The use of non-radioactive sugar-nucleotides as donor substrate, defined glycolipid precursors as acceptor substrates and of specific monoclonal anti-glycolipid antibodies for detection provides a rapid and highly specific assay for analyzing biosynthetic pathways of glycosyltransferases.     

Studies on the binding of bacteria to glycolipids. Two species of Propionibacterium apparently recognize separate epitopes on lactose of lactosylceramide

Two species of Propionibacterium were analysed regarding their binding to glycosphingolipids. Bacteria were labeled with 125I and selective interaction with glycolipids on thin-layer chromatograms was revealed by autoradiography. The carbohydrate site in common for active molecular species appeared to be lactose. The two bacteria differed, however, in the overall binding pattern on the chromatogram, probably due to recognition of separate epitopes on lactose. P. freudenreichii bound only to lactosylceramide while P. granulosum also recognized substituted lactosylceramide: Gal alpha 1----3Gal beta 1----4Glc beta Cer, GlcNAc beta 1----3Gal beta 1----4Glc beta Cer and Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc beta Cer were active, but Gal-alpha 1----4Gal beta 1----4Glc beta Cer was inactive. Also, there was an interesting dependence on ceramide structure in the case of lactosylceramide. P. freudenreichii bound to lactosylceramide with sphingosine and non-hydroxy fatty acids but not to species with sphingosine and 2-hydroxy fatty acids, phytosphingosine and non-hydroxy fatty acids or phytosphingosine and 2-hydroxy fatty acids. For P. granulosum the situation was reversed. This may be explained by an influence of ceramide structure on the presentation of the two lactose epitopes at the assay surface. These results were supported by curves from the binding of labeled bacteria to glycolipids coated in microtiter wells and in part by binding to glycolipid-coated chicken erythrocytes.     

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.     

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.     

Monoclonal antibodies directed to chemically synthesized lactogangliotetraosylceramide, a leukemia-associated antigen having a novel branching structure

Murine leukemia cells (M1), in their undifferentiated state, have been characterized by the presence of cancer-associated lactoganglio-series glycolipids, one of which was identified as lactogangliotetraosylceramide (LcGg4) having a novel branching at the II-Gal of lactosylceramide through GlcNAc beta 1----3 and GalNAc beta 1----4 linkage, as shown below (Kannagi, R., Levery, S.B., and Hakomori, S. (1984) J. Biol. Chem., 259, 8444-8451): GalNAc beta 1----4 Gal beta 1----4Glc beta 1----1Cer GlcNac beta 1----3 Since this glycolipid is a very minor component, it has been difficult to obtain enough of the purified glycolipid for the preparation of a monoclonal antibody. We developed a method to chemically synthesize this glycolipid using a lactose unit, a ceramide unit, and two hexosamine donors as synthons and made the synthetic glycolipid available as an immunogen. The two monoclonal antibodies we obtained (YI328-18 and YI328-51, both IgG3) specifically recognized the novel branching structure and had no cross-reactivity with gangliotriaosylceramide or lactotriaosylceramide. Thus, the antibodies were found to be useful probes to detect lactogangliotetraosylceramide expressed in undifferentiated M1 leukemia cells, which disappears on induced differentiation. The results of this study indicate a new strategy to establish monoclonal antibody directed to novel minor glycolipid markers or their artificially designed analogs, employing chemically synthesized glycolipid antigens.     

Identification of N-glycolylneuraminic acid-containing gangliosides of cat and sheep erythrocytes. 252Cf fission fragment ionization mass spectrometry in the analysis of glycosphingolipids

A number of gangliosides were isolated from cat and sheep erythrocytes for use in analyzing the specificity of a panel of human anti-heterophile monoclonal antibodies. The structures of these compounds were determined by a combination of different procedures, including sugar analysis, glycosidase treatment, periodate oxidation, TLC immunostaining, methylation analysis, and mass spectrometry. These methods identified the cat erythrocytes gangliosides (C1 and C2) as N-glycolylneuraminic acid (NeuGc)-containing hematosides; C1 was shown to be NeuGc alpha 2----8NeuGc alpha 2----3Gal beta I----4Glc-Cer [NeuGc)2GD3) and C2 to be NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)GD3). The two sheep gangliosides (S1 and S2) were found to be novel glycolipids based on the paragloboside sequence; S1 was identified as NeuGc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuGc)2-disialylparagloboside) and S2 as NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)-disialylparagloboside). Structural analysis of these compounds was aided by the use of 252Cf fission fragment ionization time-of-flight mass spectrometry. This method provided easily interpretable spectra on methylated derivatives which were particularly useful in determining the sialic acid composition of the gangliosides and the sequence of their disialosyl side chains.     

1H NMR studies of a biosynthetic lacto-ganglio hybrid glycosphingolipid: confirmation of structure, interpretation of "anomalous" chemical shifts, and evidence for interresidue amide-amide hydrogen bonding.

Glycosphingolipids bearing GlcNAc beta 1----3 and GalNAc beta 1----4 linked to beta-Gal of lactosylceramide (lacto-ganglio hybrids), first isolated from a murine myelogenous leukemia cell line [Kannagi, R., Levery, S. B., & Hakomori, S. (1984) J. Biol. Chem. 259, 8444-8451], have since been found as normal components of mullet roe and English sole liver. In order to clarify the biosynthetic pathways responsible for its occurrence both as a product of normal tissues and as a possible mammalian cancer-associated antigen, the lacto-ganglio hybrid core structure LcGg4Cer was synthesized from Lc3Cer using a GalNAc beta 1----4 transferase preparation from English sole liver. A preliminary characterization of the enzyme, which may be identical to the GalNAc T-1 responsible for synthesis of GM2 ganglioside, is presented. The enzymatically synthesized product was analyzed by 1- and 2-D 1H NMR spectroscopy, confirmining its primary structure as GalNAc beta 1----4-(GlcNAc beta 1----3)Gal beta 1----4Glc beta 1----1Cer. In addition to assigning all nonexchangeable glycosyl proton resonances, measurements of several properties of the amide NH protons, including chemical shift, coupling constants, exchange rates, and temperature shift coefficients, were obtained and compared to those in the simpler constituent triglycosylceramides, Lc3- and Gg3Cer. An approximate three-dimensional structure for LcGg4Cer is proposed, consistent with all data obtained, which should be useful in discussing the results of 1H NMR analysis of compounds containing this core tetrasaccharide. The structure is characterized by an unusual arrangement of terminal N-acetylhexosamine residues, resulting in a pi-H hydrogen-bonding interaction between their acetamido groups.     

Biosynthesis of terminal Gal alpha 1----3Gal beta 1----4GlcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal:N-acetyllactosaminide alpha 1----3-galactosyltransferase from calf thymus

A UDP-Gal:Gal beta 1----4GlcNAc-R alpha 1----3- and a UDP-Gal:GlcNAc-R beta 1----4-galactosyltransferase have been purified 44,000- and 101,000-fold, respectively, from a Triton X-100 extract of calf thymus by affinity chromatography on UDP-hexanolamine-Sepharose and alpha-lactalbumin-Sepharose in a yield of 25-40%. Sodium dodecyl sulfate gel electrophoresis under reducing conditions revealed a major polypeptide species with a molecular weight of 40,000 and a minor form at Mr 42,000 for the alpha 1----3-galactosyltransferase and a major polypeptide with Mr 51,000 for the beta 1----4-galactosyltransferase. Analytical gel filtration on Sephadex G-100 yielded a monomeric form for each of the galactosyltransferases with Mr 43,000 and 59,000 respectively, in addition to peaks of activity at higher molecular weights. Isoelectric focussing of the alpha 1----3-galactosyltransferase revealed a significant charge heterogeneity with forms varying in pI values between 5.0 and 6.5. Acceptor specificity studies indicated that the purified alpha 1----3-galactosyltransferase was free from contaminating galactosyltransferase activities such as those involved in the synthesis of Gal beta 1----4GlcNAc-R and Gal beta 1----3GalNAc-R sequences, the blood group B determinant, the Pk antigen, trihexosylceramide, and ganglioside GM1. The alpha 1----3-galactosyltransferase appeared to be highly active with glycoproteins, oligosaccharides, and glycolipids having a terminal Gal beta 1----4GlcNAc beta 1----unit such as asialo-alpha 1-acid glycoprotein (Km = 1.25 mM), Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3Man beta 1----4GlcNAc (Km = 0.57 mM), and paragloboside. The action of the alpha 1----3-galactosyltransferase was found to be mutually exclusive with that of the NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase from bovine colostrum. In addition alpha 1----3-fucosylation of the N-acetylglucosamine residue in the preferred disaccharide acceptor structure completely blocked galactosylation of the alpha 1----3-galactosyltransferase.     

Structure of sulfated glucuronyl glycolipids in the nervous system reacting with HNK-1 antibody and some IgM paraproteins in neuropathy

Novel sulfated glucuronic acid-containing glycolipids have been identified in the nervous system. These glycolipids are highly antigenic and share antigenic determinants with several nervous system glycoproteins, such as neural cell adhesion molecules, myelin-associated glycoprotein, and ependymins. The structure of the major antigenic glycolipid from human peripheral nerve was determined by chemical and enzymatic degradation, incorporation studies, sugar analysis after permethylation, pertrimethylsilylation, and gas liquid chromatography-mass spectrometry techniques as well as fast atom bombardment-mass spectrometry of the native antigen. The following structure was established for the major antigenic glycolipid. sulfate-3-GlcA beta(1---3)Gal beta(1----4)GlcNAc beta(1----3)Gal beta(1----4)Glc beta(1----1)-ceramide. The major fatty acids in the ceramide were 18:0, 18:1, 24:0, and 24:1, with C18-sphingenine as the long chain base.     

Structural variations of O-linked oligosaccharides present in leukosialin isolated from erythroid, myeloid, and T-lymphoid cell lines

Structures of O-linked oligosaccharides of leukosialin isolated from K562 erythroid, HL-60 promyelocytic, and HSB-2 T-lymphoid cell lines were examined. Leukosialin was isolated by specific immunoprecipitation from cells which were metabolically labeled with [3H]glucosamine, and glycopeptides were isolated after Pronase digestion. O-Linked oligosaccharides were released by alkaline borohydride treatment, and the structures of purified oligosaccharides were elucidated by specific exoglycosidase digestion, Smith degradation, and methylation anaylsis. Oligosaccharides from K562 cells were found to be GalNAcOH, Gal beta 1----3GalNAcOH, NeuNAc alpha 2----6GalNAcOH, NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH. On the other hand, oligosaccharides from HL-60 and HSB-2 cells were found to be NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAcOH, Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3)Gal beta 1----3)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3Gal beta 1----3)GalNAcOH. These results clearly indicate that leukosialin can be differently glycosylated with O-linked chains, and each erythroid or myeloid (and T-lymphoid) cell line expresses a characteristic set of O-linked oligosaccharides which differ in core structures as well as in sialylation.     

Novel blood group H glycolipid antigens exclusively expressed in blood group A and AB erythrocytes (type 3 chain H). I. Isolation and chemical characterization

A series of blood group H antigens reacting with monoclonal antibody MBrl has been found in human blood group A and AB erythrocytes, but not in O or B erythrocytes. These H antigens are clearly different from the globo-H structure (Fuc alpha 1----2Gal beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer), which was previously isolated from O erythrocytes and is also reactive with the MBrl antibody. The new series of H antigens associated with blood group A has been characterized as having TLC mobilities which approximately coincide with those of H2, H3, and H4 glycolipids. One of these A-associated H antigens, having a similar TLC mobility as the H2 glycolipid, was isolated from A erythrocytes and was characterized by 1H NMR spectroscopy, methylation analysis, and enzymatic degradation as having the structure shown below: (formula, see text). The structure represents a precursor of the repetitive A epitope attached to type 2 chain, previously called type 3 chain A (Clausen, H., Levery, S. B., Nudelman, E., Tsuchiya, S., and Hakomori, S. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1199-1203). This A-associated H structure is hereby called type 3 chain H.     

Developmentally regulated expression of cell surface carbohydrates during mouse embryogenesis

Cell surface carbohydrates undergo marked alterations during mouse embryogenesis. In preimplantation embryos, many carbohydrate markers show stage-specific expression in diverse ways. In early postimplantation embryos, certain carbohydrate markers are localized in defined regions in the embryo. Important carriers of stage-specific carbohydrates are the lactoseries structure (Gal beta 1----4GlcNAc) and the globoseries structure (Gal alpha 1----4Gal). Notably, the glycoprotein-bound large carbohydrate of poly-N-acetyllactosamine-type ([Gal beta 1----4GlcNAc beta 1----3]n) carries a number of markers preferentially expressed in early embryonic cells. These markers are of practical value in analyzing embryogenesis and cell differentiation. For example, in order to monitor in vitro differentiation of multipotential embryonal carcinoma cells, stage-specific embryonic antigen-1 (SSEA-1) and the Lotus agglutinin receptor have been used as markers of the undifferentiated cells, and the Dolichos agglutinin receptor has been used as a marker of extraembryonic endoderm cells. Developmental control of cell surface carbohydrates is attained by controlled expression of activities of key glycosyltransferases; for example, the activity of N-acetylglucosaminide alpha 1----3 fucosyltransferase is lost during in vitro differentiation of embryonal carcinoma cells to parietal endoderm cells, in parallel to the disappearance of SSEA-1. Accumulating evidence suggests that poly-N-acetyllactosamine-type glycans that are abundant in early embryonic cells are involved in cell surface recognition of these cells.     

Sialylpentaosylceramide detected with anti-GM2 monoclonal antibody. Structural characterization and complementary expression with GM2 in gastric cancer and normal gastric mucosa

The ganglioside fraction of human gastric mucosa was analyzed with a newly established anti-GM2 monoclonal antibody KM531. Using this antibody, accumulation of GM2 was observed in all of four cases of gastric carcinoma. In all ganglioside fractions extracted from normal gastric mucosa obtained from eight cases of peptic ulcer GM2 itself was not detected, but three kinds of glycolipid showing slower mobility than GM2 on thin-layer plates were detected by immunostaining with KM531. These glycolipids were assigned as NGM-1, -2, and -3. They were completely lost in all carcinoma tissues and in non-cancerous gastric mucosa from two cases of gastric cancer, and they were also not detected in the ganglioside fraction of small or large intestine. Of these glycolipids, the major one, NGM-1, was isolated from the pooled ganglioside fraction of normal gastric mucosa obtained from cases of peptic ulcer. The structure was determined by proton nuclear magnetic resonance, negative ion fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry, and treatment with exoglycosidases and mild acid hydrolysis. The structure was GalNAc beta 1----4(NeuAc alpha 2----3) Gal beta 1----4GlcNAc beta 1----3 Gal beta 1----4Glc beta 1----1Cer, which has the same terminal sequence as GM2 but has internal neolacto series structure. This epitope was previously identified as Cad blood group antigen. The decrease of this glycolipid and the increase of GM2 was considered to be a cancer-associated change in gastric mucosa.     

Structures of glycosphingolipids isolated from human granulocytes. The presence of a series of linear poly-N-acetyllactosaminylceramide and its significance in glycolipids of whole blood cells

Structures of glycolipids isolated from human granulocytes were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase treatment. All neutral glycolipids, with saccharide residues ranging from 2 to 10, were found to have linear N-acetyllactosaminyl backbones. The majority of neutral glycolipids contain one or two fucosyl residues attached to N-acetylglucosamine residues through the Fuc alpha 1----3 linkage and were reactive with the monoclonal antibody specific to Gal beta 1----4(Fuc alpha 1----3)GlcNAc, the Lex structure. Their general structure can be expressed as follows: (formula; see text) where n = 0-3. Glycolipids containing sialic acid (gangliosides) were also found to have linear N-acetyllactosaminyl backbones with sialic acid joined to this backbone by either alpha 2----3 or alpha 2----6 linkage. The gangliosides have the following general structure: (formula; see text) where n = 0-3. The ceramide was composed of sphingosine with d18:1 as the long-chain base and C16:0 (as a major component) or C24:1 (as a minor component) fatty acid. Analysis of glycolipids isolated from granulocytes, erythrocytes, and whole blood cells revealed that, among the glycolipids prepared from the whole blood cells, dihexaosylceramide, lactoneotetraosylceramide, and the above described linear lactoneo series neutral glycolipids are present in granulocytes but barely present in erythrocytes.     

Distribution of Gal alpha 1----3Gal beta 1----4GlcNAc residues on secreted mammalian glycoproteins (thyroglobulin, fibrinogen, and immunoglobulin G) as measured by a sensitive solid-phase radioimmunoassay

The study of the expression of Gal alpha 1----3Gal beta 1----4GlcNAc residues on mammalian glycoconjugates is of particular interest since as many as 1% of circulating IgG antibodies in man (the natural anti-Gal antibody) interact specifically with this carbohydrate residue. In recent studies, we have found that Gal alpha 1----3Gal beta 1----4GlcNAc residues are abundant on red cells and nucleated cells of nonprimate mammals, prosimians, and New World monkeys, but their expression is diminished in Old World monkeys, apes, and humans. In the present work, we have analyzed the expression of these residues on secreted mammalian glycoproteins. For this purpose, we have developed a radioimmunoassay (RIA) which enables the quantification of Gal alpha 1----3Gal beta 1----4GlcNAc residues on the secreted glycoproteins. Purified biotinylated anti-Gal was used as the antibody in the RIA, and bovine thyroglobulin enriched for Gal alpha 1----3Gal beta 1----4GlcNAc residues served as a solid-phase antigen. In this study, it is reported for the first time that the evolutionary pattern of Gal alpha 1----3Gal beta 1----4GlcNAc residue distribution in in vivo secreted glycoproteins is similar to that observed in membranes of cell lines and of red cells. Thyroglobulin, fibrinogen, or IgG molecules from nonprimate mammals and from New World monkeys express varying amounts of Gal alpha 1----3Gal beta 1----4GlcNAc residues ranging between 0.01 and 11 residues per molecule, whereas no such residues are present on any of these glycoproteins of human or Old World monkey origin.(ABSTRACT TRUNCATED AT 250 WORDS)