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.     

Gangliosides GM2, IV4GalNAcGM1b, and IV4GalNAcGC1a as antigens for monoclonal immunoglobulin M in neuropathy associated with gammopathy

It was previously reported that monoclonal IgM from two patients with gammopathy and neuropathy showed similar specificity by reacting with the same group of unidentified minor components in the ganglioside fractions of human nervous tissues (Ilyas, A. A., Quarles, R. H., Dalakas, M. C., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6697-6700). Enzymatic degradation, ion-exchange chromatography, and immunostaining of purified ganglioside standards on thin-layer chromatograms have now revealed that the antigenic glycolipids recognized by the IgM from these patients are gangliosides GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1Cer(GM2), GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer (IV4GalNAcGM1b), and GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4 beta Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1-Cer (IV4GalNAcGD1a). The monoclonal IgM appears to be reacting with the terminal [GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-] moiety shared by these three gangliosides and is a useful probe for detecting small amounts of GM2, IV4GalNAcGM1b, IV4GalNAcGD1a, and other gangliosides with the same terminal sugar configuration in tissues. Species distribution studies using the antibody revealed that GM2 is present in the brains and nerves of all species examined, while IV4GalNAcGM1b and IV4GalNAcGD1a exhibit some striking species specificity. GM2, but not IV4GalNAcGD1a, is enriched in purified myelin from human brain.     

Carbohydrate analysis of chicken heart glycolipids

Neutral and acidic glycolipids were extracted from chicken hearts. The neutral and acidic compounds were separated by preparative thin-layer chromatography into eight and two fractions, respectively. Total hydrolysis by mineral acid, permethylation analysis, and sequential cleavage with exoglycosidases showed the presence of glycolipids that belong to the globo- and gala-oligosaccharide series, i.e., the monohexosylceramides Glc-Cer and Gal-Cer, the dihexosylceramides Gal beta 1-4Glc-Cer and Gal alpha 1-4Gal-Cer, the tetrahexosylceramides GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer and GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal-Cer (III3GalNAc alpha-Ga3Cer) and four subfractions of the Forssman glycolipid GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer. With the notable exception of III3GalNAc alpha 1-Ga3Cer, all glycolipids with terminal GalNAc alpha 1-3GalNAc1 reacted on thin-layer chromatograms with a monoclonal anti-Forssman antibody. The major components of the acidic fraction glycolipids were characterized as the lactose-based gangliosides Glac1 (GM3) and Glac2 (GD3).     

Escherichia coli K99 binds to N-glycolylsialoparagloboside and N-glycolyl-GM3 found in piglet small intestine

Escherichia coli K12, which possess the K99 plasmid and synthesize K99 fimbriae (E. coli K99), cause severe neonatal diarrhea in piglets, calves, and lambs but not in humans. The organism binds specifically and with high affinity to only two glycolipids in piglet intestinal mucosa as demonstrated by overlaying glycolipid chromatograms with 125I-labeled bacteria. These glycolipids, which are N-glycolyl-GM3 (NeuGc alpha 2-3Gal beta 1-4Glc beta 1-1Cer) and N-glycolylsialoparagloboside (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), occur at about 13 and 0.3 micrograms per gram wet weight of mucosa, respectively. E. coli K99 grown at 18 degrees C, a temperature at which the K99 fimbriae are not expressed, do not bind to these glycolipids. Of the standard glycolipids tested in solid phase binding assays, E. coli K99 binds with highest affinity to N-glycolylsialoparagloboside, with less affinity to N-glycolyl-GM3, and with very low affinity to N-acetylsialoparagloboside. The bacteria do not bind to GM3 (NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer), GM2 (GalNAc beta 1-4[Neu-Ac alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), GM1 (Gal beta 1-3GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), or several other N-acetylsialic acid-containing gangliosides and neutral glycolipids at the levels tested. N-Glycolylsialyl residues are found in the glycoproteins and glycolipids of piglets, calves, and lambs but not in the glycoproteins and glycolipids of humans. Possibly this distribution of sialyl derivatives explains the host range of infection by the organism.     

Pseudomonas aeruginosa and Pseudomonas cepacia isolated from cystic fibrosis patients bind specifically to gangliotetraosylceramide (asialo GM1) and gangliotriaosylceramide (asialo GM2)

Pseudomonas aeruginosa infection in the lungs is a leading cause of death of patients with cystic fibrosis, yet a specific receptor that mediates adhesion of the bacteria to host tissue has not been identified. To examine the possible role of carbohydrates for bacterial adhesion, two species of Pseudomonas isolated from patients with cystic fibrosis were studied for binding to glycolipids. P. aeruginosa and P. cepacia labeled with 125I were layered on thin-layer chromatograms of separated glycolipids and bound bacteria were detected by autoradiography. Both isolates bound specifically to asialo GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) and asialo GM2 (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) but not to lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), globoside (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer), paragloboside (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), or several other glycolipids that were tested. Asialo GM1 and asialo GM2 bound the bacteria equally well, exhibiting similar binding curves in solid-phase binding assays with a detection limit of 200 ng of either glycolipid. Both isolates also did not bind to GM1, GM2, or GDla suggesting that substitution of the glycolipids with sialosyl residues prevents binding. As the Pseudomonas do not bind to lactosylceramide, the beta-N-acetylgalactosamine residue, positioned internally in asialo GM1 and terminally in asialo GM2, is probably required for binding. beta-N-Acetylgalactosamine itself, however, is not sufficient as the bacteria do not bind to globoside or to the Forssman glycolipid. These data suggest that P. aeruginosa and P. cepacia recognize at least terminal or internal GalNAc beta 1-4Gal sequences in glycolipids which may be receptors for these pathogenic bacteria.     

Molecular cloning of the human beta1,4 N-acetylgalactosaminyltransferase responsible for the biosynthesis of the Sd(a) histo-blood group antigen: the sequence predicts a very long cytoplasmic domain

The Sd(a) antigen is a carbohydrate determinant expressed on erythrocytes, the colonic mucosa and other tissues. This epitope, whose structure is Siaalpha2,3[GalNAcbeta1,4]Gal beta1,4GlcNAc, is synthesized by a beta1,4 N-acetylgalactosaminyltransferase (beta4GalNAc-T) that transfers a beta1,4-linked GalNAc to the galactose residue of an alpha2,3-sialylated chain. We have cloned from human colon carcinoma Caco2 cells a cDNA whose transfection in COS cells induces a GalNAc-T active on sialylated but not on asialylated fetuin and putatively represents the human Sd(a) beta4GalNAc-T. The cDNA predicts a 566 aa protein showing 66.6% and 39% identity with mouse CT beta4GalNAc-T and human GM2/GD2 synthase, respectively, with a typical type II glycosyltransferase organization, no potential N-glycosylation sites and a 67 aa cytoplasmic tail, which is probably the longest among the glycosyltransferases cloned to date. The gene maps in chromosome 17q23, and is composed of at least 11 exons. Exons 2-11 are homologous to exons 2-11 of the previously cloned CT beta4GalNAc-T from murine cytotoxic T lymphocytes while exons 1 of the two enzymes are totally different. The mRNA is expressed at a high level in differentiated Caco2 cells and in colonic mucosa and at a much lower level in lymphocytes and other colon cancer cell lines.     

Isolation and characterization of gangliosides from Trypanosoma brucei

Gangliosides were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC) and TLC immunostaining test. Four species of gangliosides, designated as G-1, G-2, G-3, and G-4, were separated by TLC. G-1 ganglioside had the same TLC migration rate as GM3. In contrast, G-2, G-3, and G-4 gangliosides migrated a little slower than GM1, GD1a, and GD1b, respectively. To characterize the molecular species of gangliosides from T. brucei, G-1, G-2, G-3, and G-4 gangliosides were purified and analyzed by TLC immunostaining test with monoclonal antibodies against gangliosides. G-1 ganglioside showed the reactivity to the monoclonal antibody against ganglioside GM3. G-2 was recognized by the anti-GM1 monoclonal antibody. G-3 showed reaction with the monoclonal antibody to GD1a. G-4 had the reactivity to anti-GD1b monoclonal antibody. Using 4 kinds of monoclonal antibodies, we also studied the expression of GM3, GM1, GD1a, and GD1b in T. brucei parasites. GM3, GM1, GD1a, and GD1b were detected on the cell surface of T. brucei. These results suggest that G-1, G-2, G-3, and G-4 gangliosides are GM3 (NeuAc alpha2-3Gal beta1-4Glc beta1-1Cer), GM1 (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), GD1a (NeuAc alpha2-3Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), and GD1b (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-8NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), respectively, and also that they are expressed on the cell surface of T. brucei.     

Human ovarian cancer, lymphoma spleen, and bovine milk GlcNAc:beta1,4Gal/GalNAc transferases: two molecular species in ovarian tumor and induction of GalNAcbeta1,4Glc synthesis by alpha-lactalbumin

Affinity Gel-UDP was utilized to purify GlcNAc:beta1,4Gal/GalNAc transferases (Ts) from human lymphoma spleen, ovarian tumor, and ovarian cancer sera. Mn(2+) was found to be an absolute requirement for activity. Two molecular species containing both beta1,4Gal/GalNAc-T activities were discernible when the purified ovarian tumor microsomal enzyme was subjected to Sephacryl S-100 HR column chromatography as well as native polyacylamide gel-electrophoresis. Acceptor specificity studies of the affinity-purified lymphoma spleen and ovarian tumor microsomal enzymes and the conventionally purified, as well as the cloned, bovine milk GlcNAc:beta1,4Gal-Ts using a number of synthetic acceptors showed that the beta(1,6)-linked GlcNAc moiety to alpha-GalNAc was the most efficient acceptor. As compared to the purified milk enzyme, the recombinant form exhibited sixfold GlcNAc:beta1,4 GalNAc-T activity and up to eightfold GlcNAc6SO3beta-:beta1,4Gal-T activity. Further, the recombinant enzyme catalyzed the transfer of GalNAc to the terminal beta-linked GlcNAc6SO3 moiety. Alpha-lactalbumin (alpha-LA) inhibited up to 85%, the transfer of Gal to the GlcNAc moiety linked either to Man or GlcNAc. On the contrary, alpha-LA had no significant influence on the transfer of GalNAc to the above acceptors. alpha-LA had no appreciable effect on the recombinant enzyme, except for the transfer of Gal or GalNAc to Glc. Both alpha- and beta-glucosides, as well as alpha-N-acetylglucosaminide, did not serve as acceptors.     

A new ganglioside of the lactotetraose series, GalNAc-3'-isoLM1, detected in human meconium

A monoclonal antibody produced by immunization with cells of the human glioma cell line D-54 MG reacted with ganglioside GM2. The binding epitope of the antibody was found to be GalNAc beta 1-4(NeuAc alpha 2-3)Gal. Immunological detection of glycolipid antigens on thin-layer plates with this monoclonal antibody, DMAb-1, revealed the presence of a new ganglioside. This ganglioside, co-migrating with NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer(6'-LM1) and GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-3GalNAc beta 1-4Gla beta 1-4Glc beta 1-1Cer (GalNAc-isoGM1) at chromatographic separation was isolated from human meconium. Its structure was determined by permethylation and fast atom bombardment-mass spectometry analyses. The new ganglioside was found to be a combination of the lacto and ganglio series gangliosides, and the structure found to be GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-3GlcNAc alpha 1-3Gal beta 1-4Glc beta 1-1Cer(GalNAc-3'-isoLM1).     

Mucin synthesis. Conversion of R1-beta 1-3Gal-R2 to R1-beta 1-3(GlcNAc beta 1-6)Gal-R2 and of R1-beta 1-3GalNAc-R2 to R1-beta 1-3(GlcNAc beta 1-6)GalNAc-R2 by a beta 6-N-acetylglucosaminyltransferase in pig gastric mucosa

A UDP-GlcNAc:R1-beta 1-3Gal(NAc)-R2 [GlcNAc to Gal(NAc)] beta 6-N-acetylglucosaminyltransferase activity from pig gastric mucosa microsomes catalyzes the formation of GlcNAc beta 1-3(GlcNAc beta 1-6)Gal-R from GlcNAc beta 1-3Gal-R where -R is -beta 1-3GalNAc-alpha-benzyl or -beta 1-3(GlcNAc beta 1-6)GalNAc-alpha-benzyl. This enzyme is therefore involved in the synthesis of the I antigenic determinant in mucin-type oligosaccharides. The enzyme also converts Gal beta 1-3Gal beta 1-4Glc to Gal beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc. The enzyme was stimulated by Triton X-100 at concentrations between 0 and 0.2% and was inhibited by Triton X-100 at 0.5%. There is no requirement for Mn2+ and the enzyme activity is reduced to 65% in the presence of 10 mM EDTA. Enzyme products were purified and identified by proton NMR, methylation analysis and beta-galactosidase digestion. Competition studies suggest that this pig gastric mucosal beta 6-GlcNAc-transferase activity is due to the same enzyme that converts Gal beta 1-3GalNAc-R to mucin core 2, Gal beta 1-3(GlcNAc beta 1-6)GalNAc-R, and GlcNAc beta 1-3GalNAc-R to mucin core 4, GlcNAc beta 1-3(GlcNAc beta 1-6)GalNAc-R. Substrate specificity studies indicate that the enzyme attaches GlcNAc to either Gal or GalNAc in beta (1-6) linkage, provided these residues are substituted in beta (1-3) linkage by either GlcNAc or Gal. The insertion of a GlcNAc beta 1-3 residue into Gal beta 1-3GalNAc-R to form GlcNAc beta 1-3Gal beta 1-3GalNAc-R prevents insertion of GlcNAc into GalNAc. These studies establish several novel pathways in mucin-type oligosaccharide biosynthesis.     

Molecular cloning and expression of a second chondroitin N-acetylgalactosaminyltransferase involved in the initiation and elongation of chondroitin/dermatan sulfate

We identified a novel human chondroitin N-acetylgalactosaminyltransferase, designated chondroitin GalNAcT-2 after a BLAST analysis of the GenBank(TM) data base using the sequence of a previously described human chondroitin N-acetylgalactosaminyltransferase (chondroitin GalNAcT-1) as a probe. The new cDNA sequence contained an open reading frame encoding a protein of 542 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 60% identity to that of human chondroitin GalNAcT-1. Like chondroitin GalNAcT-1, the expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which not only transferred beta1,4-N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to a polymer chondroitin representing growing chondroitin chains (beta-GalNAc transferase II activity) but also to GlcUA beta 1-3Gal beta 1-O-C(2)H(4)NHCbz, a synthetic substrate for beta-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein-linkage region of chondroitin sulfate. In contrast, the tetrasaccharide serine (GlcUA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser) derived from the linkage region, which is an inert acceptor substrate for chondroitin GalNAcT-1, served as an acceptor substrate. The coding region of this enzyme was divided into seven discrete exons, which is similar to the genomic organization of the chondroitin GalNAcT-1 gene, and was localized to chromosome 10q11.22. Northern blot analysis revealed that the chondroitin GalNAcT-2 gene exhibited a ubiquitous but differing expression in human tissues, and the expression pattern differed from that of chondroitin GalNAcT-1. Thus, we demonstrated redundancy in the chondroitin GalNAc transferases involved in the biosynthetic initiation and elongation of chondroitin sulfate, which is important for understanding the biosynthetic mechanisms leading to the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate and heparin/heparan sulfate chains.     

Evidence for the presence of two separate protein activators for the enzymic hydrolysis of GM1 and GM2 gangliosides.

Two different protein activators were isolated simultaneously from human liver for the enzymic hydrolysis of GM1 (Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-galactosidase and GM2 (GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-hexosaminidase A. The hydrolysis of GM1 is stimulated only by the GM1-specific activator which has very little effect on the hydrolysis of GM2. The same is also true for the hydrolysis of GM2. The antiserum raised against GM1 activator did not cross-react with GM2 activator and vice versa. These results suggest the presence of two different activators for the separate hydrolysis of GM1 and GM2. In connection with the enzymic hydrolysis of GM1 and GM2, we found that the hydrolysis of GM2 by human hepatic beta-N-acetylhexosaminidase A was severely inhibited by a buffer of high ionic strength, whereas no such inhibition was observed in the hydrolysis of GM1 by beta-galactosidase.     

The c-series gangliosides GT3, GT2 and GP1c are formed in rat liver Golgi by the same set of glycosyltransferases that catalyse the biosynthesis of asialo-, a- and b-series gangliosides.

Biosynthesis of the c-series gangliosides GT3, GT2 and GP1c was studied in Golgi derived from rat liver. Competition experiments show that the synthesis of ganglioside GT2 (GalNAc beta 1----4-(NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal- beta 1----4Glc beta 1----1Cer) from GT3 (NeuAc alpha 2----8NeuAc alpha 2----8-NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) seems to be catalysed by the same N-acetylgalactosaminyl-transferase (GalNAc-T), which converts GM3 (NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) to GM2 (GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1Cer). Similar competition experiments suggest moreover that the sialytransferase V (SAT V), which catalyses the synthesis of GT1a (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4- (NeuAc alpha 2----3)-Gal beta 1----4Glc beta 1----1Cer) from GD1a (NeuAc alpha-2----3Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1-Cer) appears to be identical to the enzyme that catalyses the synthesis of GP1c (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3-GalNAc beta 1----4(NeuAc alpha 2----8-NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta-1----4Glc beta 1----4Glc beta 1----1Cer) from GQ1c (NeuAc alpha 2----3Gal beta 1----3Gal-NAc beta 1----4 (NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4-Glc beta 1----1Cer).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of mammalian UDP-GalNAc:glucuronide alpha 1-4-N-acetylgalactosaminyltransferase.

We previously reported that cultured cells incubated with beta-xylosides synthesized alpha-GalNAc-capped GAG-related xylosides, GalNAc alpha GlcA beta Gal beta Gal beta Xyl beta-R and GalNAc alpha GlcA beta GalNAc beta GlcA beta Gal beta Gal beta Xyl beta-R, where R is 4-methylumbelliferyl or p-nitrophenyl (Manzi et al., 1995; Miura and Freeze, 1998). In this study, we characterized an alpha-N-acetylgalactosaminyltransferase (alpha-GalNAc-T) that probably adds the alpha-GalNAc residue to the above xylosides. Microsomes from several animal cells and mouse brain contained the enzyme activity which requires divalent cations, and has a relatively broad pH optimal range around neutral. The apparent K(m) values were in the submillimolar range for the acceptors tested, and 19 microM for UDP-GalNAc. 1H-NMR analysis of the GlcA-beta-MU acceptor product showed the GalNAc residue is transferred in alpha 1,4-linkage to the glucuronide, which is consistent with previous results reported on alpha-GalNAc-capped Xyl-MU (Manzi et al., 1995). Various artificial glucuronides were tested as acceptors to assess the influence of the aglycone. Glucuronides with a bicyclic aromatic ring, such as 4-methylumbelliferyl beta-D-glucuronide (GlcA-beta-MU) and alpha-naphthyl beta-D-glucuronide, were the best acceptors. Interestingly, a synthetic acceptor that resembles the HNK-1 carbohydrate epitope but lacking the sulfate group, GlcA beta 1,3Gal beta 1,4GlcNAc beta-O-octyl (delta SHNK-C8), was a better acceptor for alpha-GalNAc-T than the glycosaminoglycan-protein linkage region tetrasaccharyl xyloside, GlcA beta 1,3Gal beta 1,3Gal beta 1,4Xyl beta-MU. GlcA-beta-MU and delta SHNK-C8 competed for the alpha-GalNAc-T activity, suggesting that the same activity catalyzes the transfer of the GalNAc residue to both acceptors. Taken together, the results show that the alpha-GalNAc-T described here is not restricted to GAG-type oligosaccharide acceptors, but rather is a UDP-GalNAc:glucuronide alpha 1-4-N-acetylgalactosaminyltransferase.     

alpha 2,3-Sialylation of terminal GalNAc beta 1-3Gal determinants by ST3Gal II reveals the multifunctionality of the enzyme. The resulting Neu5Ac alpha 2-3GalNAc linkage is resistant to sialidases from Newcastle disease virus and Streptococcus pneumoniae

Enzymatic alpha 2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing alpha 2,3-sialylated GalNAc residues have been reported. In the present experiments, recombinant soluble alpha 2,3-sialyltransferase ST3Gal II efficiently sialylated the X(2) pentasaccharide GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, globo-N-tetraose GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and the disaccharide GalNAc beta 1-3Gal in vitro. The purified products were identified as Neu5Ac alpha 2-3GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, Neu5Ac alpha 2-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and Neu5Ac alpha 2-3GalNAc beta 1-3Gal, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enzymatic degradations, and one- and two-dimensional NMR-spectroscopy. In particular, the presence of the Neu5Ac alpha 2-3GalNAc linkage was firmly established in all three products by a long range correlation between Neu5Ac C2 and GalNAc H3 in heteronuclear multiple bond correlation spectra. Collectively, the data describe the first successful sialyltransfer reactions to the 3-position of GalNAc in any acceptor. Previously, ST3Gal II has been shown to transfer to the Gal beta 1-3GalNAc determinant. Consequently, the present data show that the enzyme is multifunctional, and could be renamed ST3Gal(NAc) II. In contrast to ST3Gal II, ST3Gal III did not transfer to the X(2) pentasaccharide. The Neu5Ac alpha 2-3GalNAc linkage of sialyl X(2) was cleaved by sialidases from Arthrobacter ureafaciens and Clostridium perfringens, but resisted the action of sialidases from Newcastle disease virus and Streptococcus pneumoniae. Therefore, the latter two enzymes cannot be used to differentiate between Neu5Ac alpha 2-3GalNAc and Neu5Ac alpha 2-6GalNAc linkages, as has been assumed previously.     

A new ganglioside showing choleragenoid-binding activity in mouse spleen

A new ganglioside showing choleragenoid-binding activity was purified from mouse spleen and characterized. From the results of sugar-composition analysis, enzymatic hydrolysis, a permethylation study, 1H-NMR spectroscopy, and negative-ion fast atom bombardment mass spectrometry, the structure of the ganglioside was determined to be as follows: Gal beta 1-3GalNAc beta 1-4Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1'ceramide 3----NeuGc alpha 2 This ganglioside contains a terminal tetrasaccharide structure identical with that of II3NeuGc alpha-Gg4Cer (GM1(NeuGc]. By means of a TLC-immunobinding assay and an enzyme-linked immunosorbent assay, the ganglioside was demonstrated to have almost the same choleragenoid-binding activity as GM1. Another ganglioside, that migrated faster than the new choleragenoid-binding ganglioside, was also purified from the same source material and identified as IV4GalNAc beta,IV3NeuGc alpha-Gg4Cer (GalNAc-GM1b(NeuGc]. Since, in the previous study, we demonstrated the existence of IV3NeuGc alpha-Gg4Cer (GM1b(NeuGc] in mouse spleen (Nakamura, K. et al. (1984) J. Biochem. 96, 949-957), the results of this study suggest that the new choleragenoid-binding ganglioside is synthesized from GM1b(NeuGc) through GalNAc-GM1b(NeuGc).     

Novel Leb-like Helicobacter pylori-binding glycosphingolipid created by the expression of human alpha-1,3/4-fucosyltransferase in FVB/N mouse stomach

The "Le(b) mouse" was established as a model for investigations of the molecular events following Le(b)-mediated adhesion of Helicobacter pylori to the gastric epithelium. By the expression of a human alpha-1,3/4-fucosyltransferase in the gastric pit cell lineage of FVB/N transgenic mice, a production of Le(b) glycoproteins in gastric pit and surface mucous cells was obtained in this "Le(b) mouse," as demonstrated by binding of monoclonal anti-Le(b) antibodies. To explore the effects of the human alpha-1,3/4-fucosyltransferase on glycosphingolipid structures, neutral glycosphingolipids were isolated from stomachs of transgenic alpha-1,3/4-fucosyltransferase-expressing mice. A glycosphingolipid recognized by BabA-expressing H. pylori was isolated and characterized by mass spectrometry and proton NMR as Fuc alpha 2Gal beta 3(Fuc alpha 4)GalNAc beta 4 Gal beta 4 Glc beta 1Cer, i.e., a novel Le(b)-like glycosphingolipid on a ganglio core. In addition, two other novel glycosphingolipids were isolated from the mouse stomach epithelium that were found to be nonbinding with regard to H. pylori. The first was a pentaglycosylceramide, GalNAc beta 3 Gal alpha 3(Fuc alpha 2)Gal beta 4 Glc beta 1Cer, in which the isoglobotetrasaccharide has been combined with Fuc alpha 2 to yield an isoglobotetraosylceramide with an internal blood group B determinant. The second one was an elongated fucosyl-gangliotetraosylceramide, GalNAc beta 3(Fuc alpha 2)Gal beta 3GalNAc beta 4Gal beta 4 Glc beta 1Cer.     

A coupled assay for UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1,6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc).

UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1,6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc) (i.e., core 2 GlcNAc-T) is a developmentally regulated enzyme of the O-linked oligosaccharide biosynthesis pathway. We have developed a coupled-enzyme assay for core 2 GlcNAc-T that is approximately 100 times more sensitive than the standard assay using UDP-[3H]GlcNAc as a sugar donor. Core 2 GlcNAc-T reactions were performed using unlabeled UDP-GlcNAc donor and Gal beta 1-3GalNAc alpha-paranitrophenyl (pNp) as acceptor. The product, Gal beta 1-3(GlcNAc beta 1-6)GalNAc alpha-pNp was then further reacted with purified bovine beta 1-4Gal-T and UDP-[3H]Gal to produce Gal beta 1-3([3H]Gal beta 1-4GlcNAc beta 1-6) GalNAc alpha-pNp, which was separated on an Ultrahydrogel HPLC column. Approximately 10% of the available GlcNAc-terminating acceptor was substituted in the Gal-T reaction, allowing 1 pmol of product to be readily detected. The increased sensitivity of the coupled assay should facilitate studies of core 2 GlcNAc-T activity where material is limiting or specific activity is low.     

Control of O-glycan synthesis: specificity and inhibition of O-glycan core 1 UDP-galactose:N-acetylgalactosamine-alpha-R beta 3-galactosyltransferase from rat liver.

The specificity of glycosyltransferases is a major control factor in the biosynthesis of O-glycans. The enzyme that synthesizes O-glycan core 1, i.e., UDP-galactose:N-acetylgalactosamine-alpha-R beta 3-galactosyltransferase (beta 3-Gal-T; EC 2.4.1.122), was partially purified from rat liver. The enzyme preparation, free of pyrophosphatases, beta 4-galactosyltransferase, beta-galactosidase, and N-acetylglucosaminyltransferase I, was used to study the specificity and inhibition of the beta 3-Gal-T. beta 3-Gal-T activity is sensitive to changes in the R-group of the GalNAc alpha-R acceptor substrate and is stimulated when the R-group is a peptide or an aromatic group. Derivatives of GalNAc alpha-benzyl were synthesized and tested as potential substrates and inhibitors. Removal or substitution of the 3-hydroxyl or removal of the 4-hydroxyl of GalNAc abolished beta 3-Gal-T activity. Compounds with modifications of the 3- or 4-hydroxyl of GalNAc alpha-benzyl did not show significant inhibition. Removal or substitution of the 6-hydroxyl of GalNAc reduced activity slightly and these derivatives acted as competitive substrates. derivatives with epoxide groups attached to the 6-position of GalNAc acted as substrates and not as inhibitors, with the exception of the photosensitive 6-O-(4,4-azo)pentyl-GalNAc alpha-benzyl, which inhibited Gal incorporation into GalNAc alpha-benzyl. The results indicate that the enzyme does not require the 6-hydroxyl of GalNAc, but needs the 3- and the axial 4-hydroxyl as essential requirements for binding and activity. In the usual biochemical O-glycan pathway, core 2 (GlcNAc beta 6[Gal beta 3] GalNAc alpha-) is formed from core 1 (Gal beta 3GalNAc-R). We have now demonstrated an alternate pathway that may be of importance in human tissues.