Structural characterization of a blood group A heptaglycosylceramide with globo-series structure. The major glycolipid based blood group A antigen of human kidney

A blood group A glycosphingolipid with the globo-series structure has been isolated from human kidney and structurally characterized. The structure was shown by mass spectrometry and proton NMR spectroscopy of the intact permethylated and permethylated-reduced derivatives together with degradation studies to be, GalNAc alpha 1----3Gal(2----1 alpha Fuc)beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1 Ceramide. This glycolipid reacts with both polyclonal and monoclonal anti-A blood group typing antisera and it is the major glycolipid based blood group A antigen present in the human kidney.     

Structure of a novel sialylated fucosyl lacto-N-norhexaosylceramide isolated from chronic myelogenous leukemia cells

A novel sialylated fucosyl glycolipid, which is present at an elevated level in chronic myelogenous leukemia cells, was isolated. The structure of this fucoganglioside was elucidated by methylation analysis, fast atom bombardment-mass spectrometry, and enzymatic degradation, followed by reaction with anti-Lex, Gal beta 1----4 (Fuc alpha 1----3) GlcNAc beta 1----, monoclonal antibody. The structure of this ganglioside was found to be: (Formula: see text). This structure is unique in that a fucose is attached to the internal N-acetylglucosamine but not to the subterminal N-acetylglucosamine. Since this glycolipid is apparently absent in normal granulocytes or acute myelogenous leukemia cells, it can be a specific marker for chronic myelogenous leukemia cells. Based on the structures of this fucoganglioside and normal granulocyte glycolipids, a biosynthetic pathway of extension, sialylation, followed by fucosylation is proposed.     

An antigen present in rat adenocarcinoma and normal colon non-epithelial stroma is a novel Forssman-like glycolipid based on isoglobotetraosylceramide

A glycolipid with blood group A activity detected in the non-epithelial stroma of normal rat colon but not in epithelial cells (Hansson, G.C., Karlsson, K.-A., and Thurin, J. (1984) Biochim. Biophys. Acta 792, 281-292), was purified to homogeneity from normal rat colon and rat colon adenocarcinoma. Mass spectrometry and 1H-NMR spectroscopy of the intact permethylated derivative and gas chromatography after degradation revealed the structure GalNAc alpha 1----3GAINAc beta 1----3Gal alpha 1----3Gal beta 1----4Glc beta 1----1Cer, with the predominant ceramide containing sphingosine and non-hydroxylated 24:0 fatty acid. This identifies this glycolipid as a novel Forssman-like glycolipid, which is a tumor-associated antigen by definition, since it is not present in the normal rat large intestinal epithelium cells but in rat adenocarcinoma derived from these cells.     

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.     

Characterization of a glycosphingolipid antigen defined by the monoclonal antibody MBr1 expressed in normal and neoplastic epithelial cells of human mammary gland

The antigen defined by a monoclonal antibody, MBr1, was found to be expressed in normal human mammary gland epithelia and human mammary carcinoma cells (Ménard, S., Tagliabue, E., Canevari, S., Fossati, G., and Colnaghi, M. I. (1983) Cancer Res. 43, 1295-1300). The antigen has been isolated from breast cancer cell line MCF-7, which was used as immunogen, and its structure was determined by methylation analysis, NMR spectroscopy, direct probe mass spectrometry, and enzymatic degradation as identified below. Fuc alpha 1----2Gal beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer The antibody cross-reacted weakly with fucosylasialo-GM1 (IV2FucGg4), which shares the same terminal sequence, Fuc alpha 1----2Gal beta 1----3GalNAc, with this antigen. However, various other structures, including lacto-series H structure (Fuc alpha 1----2 Gal beta 1----4/or 3GlcNAc beta 1----3Gal), did not show any reactivity with this antibody. Therefore, this antigen represents a blood group H antigen with a globo-series structure which is abundant in human teratocarcinoma (Kannagi, R., Levery, S. B., Ishigami, F., Hakomori, S., Shevinsky, L. H., Knowles, B. B., and Solter, D. (1983) J. Biol. Chem. 258, 8934-8942), although its presence must be limited in normal adult human tissue.     

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.     

Lewis blood group antigens defined by monoclonal anti-colon carcinoma antibodies

Monoclonal antibodies directed against human cancer cells were prepared by the murine hybridoma technique. These antibodies detect Lewis blood group antigens as determined by indirect solid-phase radioimmunoassay, hapten inhibition studies, and chromatogram binding assay. One monoclonal antibody is specific for the Lea terminal carbohydrate of Gal beta 1----3Glc NAc(4----1 alpha Fuc) beta 1----3LacCer. Five monoclonal antibodies react with the Leb terminal carbohydrate sequence of Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer, and four of these antibodies are highly specific for this glycolipid and do not react with other similar di- and monofucosylated glycolipids. One of the anti-Leb antibodies cross-reacts with blood group H glycolipid and has binding properties similar to those of the previously described antibody NS-10-17 [M. Brockhaus, J. L. Magnani, M. Blaszczyk, Z. Steplewski, H. Koprowski, K.-A. Karlsson, G. Larson, and V. Ginsburg (1981) J. Biol. Chem. 256, 13223-13225]. Two antibodies react with both the Lea and Leb antigens, though both bind preferentially to Leb.     

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.     

Hybrid type glycolipids (lacto-ganglio series) with a novel branched structure. Their presence in undifferentiated murine leukemia cells and their dependence on differentiation

The structures of these glycolipids are hybrids of the lacto and ganglio series, which are characterized by the presence of GlcNAc beta 1----3 and GalNAc beta 1----4 linked to the Gal residue of Gal beta 1----4Glc beta 1----1Cer. This new hybrid series can be designated as "lacto-ganglio series." These glycolipids are present in undifferentiated murine leukemia cells. Their concentration declines with differentiation and they are virtually absent in differentiated M1+ cells, suggesting that lacto-ganglio structures could be markers of undifferentiated, malignant myeloid cells.     

Structure of a triphosphonopentaosylceramide containing 4-O-methyl-N-acetylglucosamine from the skin of the sea hare, Aplysia kurodai

A novel phosphonoglycosphingolipid named SGL-I containing 3 mol of 2-aminoethylphosphonate residues was isolated from the skin of a sea gastropod, Aplysia kurodai. The saccharide moiety of the glycolipid was characterized as 4-O-methyl-GlcNAc alpha 1----4GalNAc alpha-1----3 [6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2] (2-aminoethylphosphonyl----6)Gal beta 1----4(2-aminoethylphosphonyl----6) Glc beta 1----1-ceramide. The major aliphatic components of the ceramide portion were palmitic acid, stearic acid, octadeca-4-sphingenine, and anteisononadeca-4-sphingenine. This glycolipid is unique in containing 4-O-methyl-N-acetylglucosamine and 3 mol of 2-aminoethylphosphonate residues, one of which is attached to C-6 of glucose.     

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 characterization of a novel 2-aminoethylphosphonyl-glycosphingolipid from the sea hare, Aplysia kurodai

A novel phosphonoglycosphingolipid named SGL-I' containing 1 mol of 2-aminoethylphosphonate residue was isolated from the skin of Aplysia kurodai using two silicic acid chromatography systems. Data obtained on methanolysis, permethylation, mild acid hydrolysis, and hydrogen fluoride treatment combined with thin-layer chromatography, gas liquid chromatography, gas chromatography-mass spectrometry, and proton magnetic resonance spectrometry showed that this glycolipid was 3-O-MeGal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2]Gal beta 1----4Glc beta 1----1Ceramide. Palmitic acid, octadeca-4-sphingenine and anteiso-nonadeca-4-sphingenine are its major aliphatic components. The new glycolipid has essentially the same structure as another major phosphonoglycosphingolipid in the skin of Aplysia, SGL-II, that contains 2 mol of 2-aminoethylphosphonate residue, suggesting a metabolic relationship between the two.     

Novel isoglobo-neolacto-series hybrid glycolipid detected by a monoclonal antibody is a rat colon tumor-associated antigen

Isoglobotetraosylceramide (GalNAc(beta 1-3)Gal(alpha 1-3)Gal(beta 1-4)Glc (beta 1-1)Cer), the major glycolipid species in dimethylhydrazine-induced rat tumors of colorectal origin, was not detected in epithelial cells of normal colon but was present in the non-epithelial stroma and could be extracted from each of nine tumors studied. Monoclonal antibodies produced against isoglobotetraosylceramide detected this and another novel rat tumor-associated glycolipid not present in epithelial cells nor in non-epithelial stroma of normal rat colon (Brodin, T., Thurin, J., Str?mberg, N., Karlsson, K.-A. and Sj?gren, H.O. (1985) Eur. J. Immunol. 16, 951-956). This novel glycolipid was present in 8/9 of the studied tumors and was also present in two in vitro cell clones. These were originally obtained from a W49/T4 colon tumor isograft. The novel glycolipid was characterized by mass spectrometry, 1H-NMR, and methylation analysis as a hybrid between the isoglobo- and neolacto-series, with the structure GalNAc(beta 1-3)Gal(alpha 1-3)Gal(beta 1-4)GlcNA(beta 1-3)Gal (beta 1-4)Glc(beta 1-1)Cer.     

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.     

Purification and characterization of the epitectin from human laryngeal carcinoma cells

The purification and partial characterization of epitectin (previously called Ca antigen) from a human cancer cell line is described. This glycoprotein, which is expressed on a wide range of human tumors and certain specialized normal epithelia, can be detected using monoclonal antibodies, Ca1, Ca2, and Ca3. The purified glycoprotein had a high density (1.40 g/ml) on isopycnic centrifugation indicating a high carbohydrate content. The molecular mass of epitectin as determined by size-exclusion chromatography ranged from 1.0 to 1.5 x 10(6) daltons. However, the purified epitectin gave two bands of apparent molecular weight 390,000 and 350,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric points of epitectin and asialoepitectin were found to be 5.3-5.4 and 6.8, respectively. The oligosaccharides were isolated from metabolically labeled epitectin by alkaline borohydride treatment and their structures established based on high performance liquid chromatography and paper electrophoretic migration, sugar composition, the results of sequential exoglycosidase treatment, periodate oxidation, and methylation analysis. The structures of the three major fractions, which together account for about 80% of the radioactivity, were assigned as NeuNAc alpha 2----3Gal beta 1----(NeuNAc alpha 2----6)3GalNAc(OH), NeuNAc alpha 2----3Gal beta 1----3GalNAc(OH), and Gal beta 1----3 GalNAc(OH). The structures of the minor fractions were tentatively assigned as NeuNAc----Gal(NeuNAc----Gal----GlcNAc)----GalNAc(OH), Gal beta 1----(NeuNAc alpha 2----6)3GalNAc(OH), NeuNAc alpha 2----6GalNAc(OH), and GalNAc(OH). It is proposed that the protein sequence and/or the distribution of the saccharides on the protein core are the determinants on epitectin that are recognized by the Ca antibodies.     

Biosynthesis in vitro of a globoside containing a 2-acetamido-2-deoxy-beta-D-galactopyranosyl group (1----3)-linked and Forssman glycolipid by two N-acetylgalactosaminyltransferases from chemically transformed guinea pig cells

Two N-acetylgalactosaminyltransferase activities (GalNAcT-2 and GalNAcT-3) have been characterized in chemically transformed, cultured guinea-pig cell lines (104C1 and 106B). Line 104C1 is a benz[a]pyrene-transformed tumorigenic variant, whereas line 106B is a 7,12-dimethylbenz[a]anthracene-transformed nontumorigenic variant obtained from fetal guinea-pig cells at 43 days of gestation. The GalNAcT-2 (UDP-GalNAc:GbOse3Cer beta-N-acetylgalactosaminyltransferase) isolated from both 104C1 and 106B cells catalyzed the transfer of Gal-NAc from UDP-GalNAc to the 3H-labeled terminal galactose group of Gb3 [( 6-3H]Gal alpha 1----4Gal beta 1----4Glc----Cer). The 3H-labeled globoside was purified and then subjected to exhaustive methylation. After acetolysis, the partially methylated sugars were separated by two-dimensional, thin-layer chromatography. 3H-Label was detected in two major areas, 2,4,6-tri-O-Me-Gal (40%) and 2,3,4,6-tetra-O-Me-Gal (46%). In a separate experiment, 80% of the GalNAc was released when labeled GbOse4Cer [( 3H]GalNAc----Gal alpha 1----4Gal beta 1----4Glc----Cer) was treated with purified clam beta-hexosaminidase. The present results establish the formation of a beta-D-GalpNAc-(1----3) linkage in the terminal region of the biosynthesized globoside. GalNAcT-3 activity (UDP-GalNAc:GbOse4Cer alpha-GalNAc-transferase), which catalyzes the transfer of GalNAc from UDP-[14C]- or -[3H]GalNAc to GbOse4Cer (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc----Cer), was three times higher in 106B cells than in 104C1 cells. The isolated, purified radioactive product formed an immunoprecipitin line against rabbit anti-Forssman antibody.     

Structure of a glycolipid reacting with monoclonal IgM in neuropathy and with HNK-1

An acidic glycolipid antigen that reacts with monoclonal IgM in patients with demyelinating neuropathy and with the mouse monoclonal antibody, HNK-1, was purified from human peripheral nerves. This lipid sharing antigenic determinants with the myelin-associated glycoprotein was shown to be an unusual glucuronic acid-containing sulfated glycosphingolipid with five sugars, but without sialic acid. Mild acid methanolysis converted the GlcUA to its methyl ester, removed the acidic sulfate group and abolished the antigenicity. Results from chemical, enzymatic, infrared, and mass spectral analysis suggested the following structure with a sulfate in a position that remains to be determined: GlcUA beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1 ceramide.     

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.     

Localization of UDP-GalNAc:NeuAc alpha 2,3Gal-R beta 1,4(GalNAc to Gal)N-acetylgalactosaminyltransferase in human stomach. Enzymatic synthesis of a fundic gland-specific ganglioside and GM2.

A glycolipid detected in human gastric mucosa with anti-GM2 monoclonal antibody was characterized to be GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4GlcNAc beta 1-3Gal 1-4Glc-Cer (NGM-1), which was lost in gastric cancer tissue with complementary increase of GM2 sharing the same terminal carbohydrate structure as NGM-1 (Dohi, T., Ohta, S., Hanai, N., Yamaguchi, K., and Oshima, M. (1990) J. Biol. Chem. 265, 7880-7885). The study on differential expression of NGM-1 in gastric fundic mucosa, pyloric mucosa, gastric cancer, and various other tissues indicated that NGM-1 existed specifically in fundic mucosa. The content of GM3 and sialylparagloboside (SPG), which are the substrates for the synthesis of GM2 and NGM-1, respectively, were not significantly different in these tissues. Therefore, the presence of two kinds of beta 1,4GalNAc transferases having different substrate specificity was considered to be critical for the expression of NGM-1 and GM2. The activity of beta 1,4GalNAc transferase which synthesizes GM2 or NGM-1 was determined by detecting the products with specific monoclonal antibodies. The activity of beta 1,4GalNAc transfer to SPG was high in fundic mucosa, while it was absent in pyloric mucosa or cancer. On the other hand, the increased activity of beta 1,4GalNAc transfer to GM3 was observed in cancer tissues and cancer cell lines which were rich in GM2. Our conclusion is that the limited expression of NGM-1 in fundic mucosa and the increase of GM2 in cancer are attributed to two types of beta 1,4GalNAc transferases localized in each region with different substrate specificity; the one in fundic mucosa transfers GalNAc to SPG but not to GM3, and the other one enhanced in cancer transfers GalNAc to GM3 but not to SPG.     

Purification and enzymatic characterization of CMP-sialic acid: beta-galactosyl1----3-N-acetylgalactosaminide alpha 2----3-sialyltransferase from human placenta

A CMP-NeuAc:Gal beta 1----3GalNAc-R alpha 2----3-sialyltransferase has been purified over 20,000-fold from a Triton X-100 extract of human placenta by affinity chromatography on concanavalin A-Sepharose and CDP-hexanolamine-Sepharose in a yield of 10%. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions revealed that the enzyme consists of a major polypeptide species with a molecular weight of 41,000 and some minor forms with molecular weights of 40,000, 43,000, and 65,000, respectively, which can be resolved partially by gel filtration on Sephadex G-100. Isoelectric focusing revealed that the enzyme occurs in a major and a minor charged form with pI values of 5.0-5.5 and 6.0, respectively. Acceptor specificity studies indicated that the enzyme catalyzes the incorporation of sialic acid from CMP-NeuAc into glycoproteins, glycolipids, and oligosaccharides which possess a terminal Gal beta----3GalNAc unit. Analysis of the structure of the product chain by high-pressure liquid chromatography and thin layer chromatography as well as methylation analysis revealed that a NeuAc alpha 2----3Gal beta 1----3GalNAc sequence is elaborated. The best glycoprotein acceptors are antifreeze glycoprotein and porcine submaxillary asialo/afucomucin. The disaccharide Gal beta 1----3GalNAc-Thr shows values for Km and V which are close to those of the latter glycoprotein. Lactose as well as oligosaccharides in which galactose is linked beta 1----3 or beta 1----4 to N-acetylglucosamine are less efficient acceptors. Of the glycolipids tested only gangliosides GM1 and GD1b served as an acceptor. The enzyme does not show an absolute aglycon specificity, and attaches sialic acid regardless the anomeric configuration of the N-acetylgalactosaminyl residue in the accepting Gal beta 1----3GalNAc unit. By use of specific acceptor substrates it could be demonstrated that the purified enzyme is free from other known sialyltransferase activities. Studies with rabbit antibodies raised against a partially purified sialyltransferase preparation indicated that the enzyme is immunologically unrelated to a Gal beta 1----4GlcNAc-R alpha 2----3-sialyltransferase, which previously had been identified in human placenta (Van den Eijnden, D.H., and Schiphorst, W. E. C. M. (1981) J. Biol. Chem. 256, 3159-3162). Initial-rate kinetic studies suggest that the sialyltransferase operates through a mechanism involving a ternary complex of enzyme, sugar donor, and acceptor. This is the first report on the extensive purification and characterization of a sialyltransferase from a human tissue.