Toshie Iwai

Molecular Cloning and Characterization of a Novel UDP-GlcNAc:GalNAc-peptide β1,3-N-Acetylglucosaminyltransferase (β3Gn-T6), an Enzyme Synthesizing the Core 3 Structure of O-Glycans

The core 3 structure of theO-glycan, GlcNAcβ1–3GalNAcα1-serine/threonine, an important precursor in the biosynthesis of mucin-type glycoproteins, is synthesized by UDP-N-acetylglucosamine:GalNAc-peptide β1,3-N- acetylglucosaminyltransferase (β3Gn-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 β3Gn-T family was found in one of the data bases. We cloned a complementary DNA of this gene and named it β3Gn-T6. The putative amino acid sequence of β3Gn-T6 retains the β3Gn-T motifs and is predicted to comprise a typical type II membrane protein. The soluble form of β3Gn-T6 expressed in insect cells showed β3Gn-T activity toward GalNAcα-p-nitrophenyl and GalNAcα1-serine/threonine. The β1,3-linkage between GlcNAc and GalNAc of the enzyme reaction product was confirmed by high performance liquid chromatography and NMR analyses. β3Gn-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 β3Gn-T6 transcript was restricted in its distribution, mainly to the stomach, colon, and small intestine. We concluded that β3Gn-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.

A novel β1,3-N-acetylglucosaminyltransferase (β3Gn-T8), which synthesizes poly-N-acetyllactosamine, is dramatically upregulated in colon cancer

A new member of the UDP‐N‐acetylglucosamine: β‐galactose β1,3‐N‐acetylglucosaminyltransferase (β3Gn‐T) family having the β3‐glycosyltransferase motifs was identified using an in silico method. This novel β3Gn‐T was cloned from a human colon cancer cell line and named β3Gn‐T8 based on its position in a phylogenetic tree and enzymatic activity. β3Gn‐T8 transfers GlcNAc to the non‐reducing terminus of the Galβ1–4GlcNAc of tetraantennary N‐glycan in vitro. HCT15 cells transfected with β3Gn‐T8 cDNA showed an increase in reactivity to both LEA and PHA‐L4 in a flow cytometric analysis. These results indicated that β3Gn‐T8 is involved in the biosynthesis of poly‐N‐acetyllactosamine chains on tetraantennary (β1,6‐branched) N‐glycan. In most of the colorectal cancer tissues examined, the level of β3Gn‐T8 transcript was significantly higher than in normal tissue. β3Gn‐T8 could be an enzyme involved in the synthesis of poly‐N‐acetyllactosamine on β1–6 branched N‐glycans in colon cancer.

Molecular cloning and characterization of β1,4‐N‐acetylgalactosaminyltransferases IV synthesizing N,N′‐diacetyllactosediamine1

A sequence highly homologous to β1,4‐N‐acetylgalactosaminyltransferase III (β4GalNAc‐T3) was found in a database of human expressed sequence tags. The full‐length open reading frame of the gene, β4GalNAc‐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 β4GalNAc‐T3. The recombinant enzyme transferred N‐acetylgalactosamine to N‐acetylglucosamine‐β‐benzyl with a β1,4‐linkage to form N,N′‐diacetyllactosediamine as did β4GalNAc‐T3. In specificity toward oligosaccharide acceptor substrates, it was quite similar to β4GalNAc‐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.

Regulation of Glycosyltransferases for O-Glycan Core Structure in Cancer and Their Relations to Metastasis

Synthesis of many O-linked carbohydrate chains (O-glycan) is initiated by transferring GalNAc to the Ser/Thr residue in an amino acid sequence by polypeptide GalNAc transferase (pp-GalNAc-T) (Cheng et al. 2004). These O-glycans are called mucin-type carbohydrate chains, and most of them are found on glycoproteins called mucin. Mucin, which is the major constituent of mucus glycoproteins, not only protects the mucosal membrane from invasion of viruses and chemical substances but also is involved in various biological phenomena including cell adhesion, signal transduction, inflammation, and metastasis.