Apical Golgi localization of N,N'-diacetyllactosediamine synthase, beta4GalNAc-T3, is responsible for LacdiNAc expression on gastric mucosa.

beta1,4-N-acetylgalactosaminyltransferase III (beta4GalNAc-T3), which was recently cloned and identified, exhibits GalNAc transferase activity toward a GlcNAcbeta residue with beta1,4-linkage, forming the N,N'-diacetyllactosediamine, GalNAcbeta1,4GlcNAc (LacdiNAc or LDN). Though LacdiNAc has not been found in the gastric mucosa, a large amount of transcript was detected in our previous study. To increase our knowledge of beta4GalNAc-T3 expression and its product LacdiNAc, we examined the exact localization of beta4GalNAc-T3 in human gastric mucosa using a newly developed antibody, monoclonal antibody (mAb) K1356. This antibody specifically detected the enzyme that transfected the beta4GalNAc-T3 gene into MKN45 cells, and the terminal betaGalNAc epitope yielded on the cell surface was recognized by a lectin, Wisteria floribunda agglutinin (WFA). beta4GalNAc-T3 was localized in the supra-nuclear region of surface mucous cells in gastric mucosa, and WFA positively stained the mucins secreted by the cells. In contrast, in the cells of the glandular compartment in the fundic glands and a few cells in the pyloric glands, beta4GalNAc-T3 was observed in the basolateral position of the nucleus, where no WFA reactivity was detected. The anti-Tn (GalNAcalpha-O-Ser/Thr) antibody staining did not overlap with the WFA staining. By measuring the binding activity of WFA using automated frontal affinity chromatography (FAC), we found WFA to bind most strongly LacdiNAc among the sugar chains examined. Neither beta4GalNAc-T3 nor WFA-positive staining was detected in intestinal metaplastic cells. These results suggest that the supra-nuclear expression of beta4GalNAc-T3 is essential for the formation of LacdiNAc on the surface mucous cells and that LacdiNAc and beta4GalNAc-T3 are novel differentiation markers of surface mucous cells in the gastric mucosa.

Core 3 synthase is down-regulated in colon carcinoma and profoundly suppresses the metastatic potential of carcinoma cells.

The core 3 structure of the O-glycan, GlcNAcbeta1-3GalNAcalpha1-Ser/Thr, an important precursor in the biosynthesis of mucin-type glycoproteins, is synthesized by beta1,3-N-acetylglucosaminyltransferase 6 (beta3Gn-T6; core 3 synthase). We generated an anti-beta3Gn-T6 mAb (G8-144 mAb) and performed immunohistochemical analyses. In normal stomach and colon, beta3Gn-T6 was strongly expressed in the Golgi region of epithelia. In contrast, its expression was markedly down-regulated in gastric and colorectal carcinomas. Tissue specimens from a familial adenomatous polyposis patient showed a clear correlation between the down-regulation of beta3Gn-T6 expression and the degree of dysplasia/neoplasia. In vitro, the level of beta3Gn-T6 transcript was increased according to the differentiation of Caco-2 cells. These results suggested that the expression of beta3Gn-T6 is closely regulated during differentiation and dedifferentiation. beta3Gn-T6 would be a useful marker for distinguishing between benign adenomas and premalignant lesions. HT1080 FP-10 cells stably transfected with the beta3Gn-T6 gene showed a decrease in the core 1 structure, Galbeta1,3GalNAcalpha1-Ser/Thr, probably due to competition between the core 1 synthase and core 3 synthase. The migration activity of the transfectants was markedly lower than that of mock transfectants in vitro, and lung metastasis after i.v. injection of the transfectants into nude mice was significantly suppressed. These findings indicated that the core structures of O-glycans are profoundly involved in the metastatic capacity of cancer cells.

A novel beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T8), which synthesizes poly-N-acetyllactosamine, is dramatically upregulated in colon cancer.

A new member of the UDP-N-acetylglucosamine: beta-galactose beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T) family having the beta3-glycosyltransferase motifs was identified using an in silico method. This novel beta3Gn-T was cloned from a human colon cancer cell line and named beta3Gn-T8 based on its position in a phylogenetic tree and enzymatic activity. Beta3Gn-T8 transfers GlcNAc to the non-reducing terminus of the Galbeta1-4GlcNAc of tetraantennary N-glycan in vitro. HCT15 cells transfected with beta3Gn-T8 cDNA showed an increase in reactivity to both LEA and PHA-L4 in a flow cytometric analysis. These results indicated that beta3Gn-T8 is involved in the biosynthesis of poly-N-acetyllactosamine chains on tetraantennary (beta1,6-branched) N-glycan. In most of the colorectal cancer tissues examined, the level of beta3Gn-T8 transcript was significantly higher than in normal tissue. Beta3Gn-T8 could be an enzyme involved in the synthesis of poly-N-acetyllactosamine on beta1-6 branched N-glycans in colon cancer.

Molecular cloning and characterization of beta1,4-N-acetylgalactosaminyltransferases IV synthesizing N,N'-diacetyllactosediamine.

A sequence highly homologous to beta1,4-N-acetylgalactosaminyltransferase III (beta4GalNAc-T3) was found in a database of human expressed sequence tags. The full-length open reading frame of the gene, beta4GalNAc-T4 (GenBank accession number AB089939), was cloned using the 5' rapid amplification of cDNA ends method. It encodes a typical type II transmembrane protein of 1039 amino acids having 42.6% identity with beta4GalNAc-T3. The recombinant enzyme transferred N-acetylgalactosamine to N-acetylglucosamine-beta-benzyl with a beta1,4-linkage to form N,N'-diacetyllactosediamine as did beta4GalNAc-T3. In specificity toward oligosaccharide acceptor substrates, it was quite similar to beta4GalNAc-T3 in vitro, however, the tissue distributions of the two enzymes were quite different. These results indicated that the two enzymes have similar roles in different tissues.

A novel I-branching beta-1,6-N-acetylglucosaminyltransferase involved in human blood group I antigen expression.

The human blood group i and I antigens are determined by linear and branched poly-N-acetyllactosamine structures, respectively. In erythrocytes, the fetal i antigen is converted to the adult I antigen by I-branching beta-1,6-N-acetylglucosaminyltransferase (IGnT) during development. Dysfunction of the I-branching enzyme may result in the adult i phenotype in erythrocytes. However, the I gene responsible for blood group I antigen has not been fully confirmed. We report here a novel human I-branching enzyme, designated IGnT3. The genes for IGnT1 (reported in 1993), IGnT2 (also presented in this study), and IGnT3 consist of 3 exons and share the second and third exons. Bone marrow cells preferentially expressed IGnT3 transcript. During erythroid differentiation using CD34(+) cells, IGnT3 was markedly up-regulated with concomitant decrease in IGnT1/2. Moreover, reticulocytes expressed the IGnT3 transcript, but IGnT1/2 was below detectable levels. By molecular genetic analyses of an adult i pedigree, individuals with the adult i phenotype were revealed to have heterozygous alleles with mutations in exon 2 (1006G>A; Gly336Arg) and exon 3 (1049G>A; Gly350Glu), respectively, of the IGnT3 gene. Chinese hamster ovary (CHO) cells transfected with each mutated IGnT3 cDNA failed to express I antigen. These findings indicate that the expression of the blood group I antigen in erythrocytes is determined by a novel IGnT3, not by IGnT1 or IGnT2.

Molecular cloning and characterization of a novel UDP-Gal:GalNAc(alpha) peptide beta 1,3-galactosyltransferase (C1Gal-T2), an enzyme synthesizing a core 1 structure of O-glycan.

Recently, a UDP-Gal:GalNAc(alpha) peptide beta1,3-galactosyltransferase (core 1 synthase 1; C1Gal-T1) has been purified from rat liver and its complementary DNA cloned from several species. We isolated a second candidate for core 1 synthase from a Colo205 cDNA library and named it C1Gal-T2. The deduced amino acid sequence of C1Gal-T2, having 26% homology to C1Gal-T1, showed a topology typical of a type II membrane protein. Real time PCR analysis revealed that the expression of C1Gal-T2 transcripts was widespread in many tissues and of relatively high level in salivary gland, stomach, small intestine, kidney, testis, thymus, and spleen. LSC cells, having no core 1 synthase activity, were transfected stably with the C1Gal-T2 gene. Their microsome fraction showed beta1,3-galactosyltransferase activity toward GalNAc-alpha-para-nitrophenyl and GalNAc(alpha)1 peptides resulting in the synthesis of the core 1 structure. The core 1 synthesizing activity of C1Gal-T2 was also determined by flow cytometry and lectin blotting using the LSC cells stably expressing C1Gal-T2. Finally, LSC cells, and Jurkat cells that also lack the core 1 synthase activity, were found to have null alleles of C1Gal-T2. These results indicated that C1Gal-T2 is the second candidate for core 1 synthase that plays an important role in synthesizing O-glycans in digestive organs.

Molecular cloning and characterization of a novel UDP-GlcNAc:GalNAc-peptide beta1,3-N-acetylglucosaminyltransferase (beta 3Gn-T6), an enzyme synthesizing the core 3 structure of O-glycans.

The core 3 structure of the O-glycan, GlcNAcbeta1-3GalNAcalpha1-serine/threonine, an important precursor in the biosynthesis of mucin-type glycoproteins, is synthesized by UDP-N-acetylglucosamine:GalNAc-peptide beta1,3-N- acetylglucosaminyltransferase (beta3Gn-T; core 3 synthase). The core 3 structure is restricted in its occurrence to mucins from specific tissues such as the stomach, small intestine, and colon. A partial sequence encoding a novel member of the human beta3Gn-T family was found in one of the data bases. We cloned a complementary DNA of this gene and named it beta3Gn-T6. The putative amino acid sequence of beta3Gn-T6 retains the beta3Gn-T motifs and is predicted to comprise a typical type II membrane protein. The soluble form of beta3Gn-T6 expressed in insect cells showed beta3Gn-T activity toward GalNAcalpha-p-nitrophenyl and GalNAcalpha1-serine/threonine. The beta1,3-linkage between GlcNAc and GalNAc of the enzyme reaction product was confirmed by high performance liquid chromatography and NMR analyses. beta3Gn-T6 effectively transferred a GlcNAc to the GalNAc residue on MUC1 mucin, resulting in the synthesis of a core 3 structure. Real time PCR analysis revealed that the beta3Gn-T6 transcript was restricted in its distribution, mainly to the stomach, colon, and small intestine. We concluded that beta3Gn-T6 is the most logical candidate for the core 3 synthase, which plays an important role in the synthesis of mucin-type O-glycans in digestive organs.