Hiroko Iwasaki

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.

α1,3‐Fucoslytransferase IX (Fuc‐TIX) is very highly conserved between human and mouse; molecular cloning, characterization and tissue distribution of human Fuc‐TIX

The amino acid sequence of Fuc‐TIX is very highly conserved between mouse and human. The number of non‐synonymous nucleotide substitutions of the Fuc‐TIX gene between human and mouse was strikingly low, and almost equivalent to that of the α‐actin gene. This indicates that Fuc‐TIX is under a strong selective pressure of preservation during evolution. The human Fuc‐TIX (hFuc‐TIX) showed a unique characteristics, i.e. hFuc‐TIX was not activated by Mn2+ and Co2+, whereas hFuc‐TIV and hFuc‐TVI were activated by the cations. The hFuc‐TIX transcripts were abundantly expressed in brain and stomach, and interestingly were detected in spleen and peripheral blood leukocytes.

Characterization of a novel human UDP-GalNAc transferase, pp-GalNAc-T15

A novel member of the human UDP‐N‐acetyl‐D‐galactosamine:polypeptide N‐acetylgalactosaminyltransferase (pp‐GalNAc‐T) gene family was cloned as a homolog of human pp‐GalNAc‐T7, and designated pp‐GalNAc‐T10. pp‐GalNAc‐T10 transcript was found in the small intestine, stomach, pancreas, ovary, thyroid gland and spleen. In a polypeptide GalNAc‐transfer assay, recombinant pp‐GalNAc‐T10 transferred GalNAc onto a panel of mucin‐derived peptide substrates. Furthermore, pp‐GalNAc‐T10 demonstrated strong transferase activity with glycopeptide substrates.

N-acetylglucosaminyltransferase IX acts on the GlcNAcβ1,2-Manα1-Ser/Thr moiety, forming a 2,6-branched structure in brain O-mannosyl glycan

Mammals contain O-linked mannose residues with 2-mono- and 2,6-di-substitutions by GlcNAc in brain glycoproteins. It has been demonstrated that the transfer of GlcNAc to the 2-OH position of the mannose residue is catalyzed by the enzyme, protein O-mannose β1,2-N-acetylglucosaminyltransferase (POMGnT1), but the enzymatic basis of the transfer to the 6-OH position is unknown. We recently reported on a brain-specific β1,6-N-acetylglucosaminyltransferase, GnT-IX, that catalyzes the transfer of GlcNAc to the 6-OH position of the mannose residue of GlcNAcβ1,2-Manα on both the α1,3- and α1,6-linked mannose arms in the core structure of N-glycan (Inamori, K., Endo, T., Ide, Y., Fujii, S., Gu, J., Honke, K., and Taniguchi, N. (2003) J. Biol. Chem. 278, 43102–43109). Here we examined the issue of whether GnT-IX is able to act on the same sequence of the GlcNAcβ1,2-Manα in O-mannosyl glycan. Using three synthetic Ser-linked mannose-containing saccharides, Manα1-Ser, GlcNAcβ1,2-Manα1-Ser, and Galβ1,4-GlcNAcβ1,2-Manα1-Ser as acceptor substrates, the findings show that 14C-labeled GlcNAc was incorporated only into GlcNAcβ1,2-Manα1-Ser after separation by thin layer chromatography. To simplify the assay, high performance liquid chromatography was employed, using a fluorescence-labeled acceptor substrate GlcNAcβ1,2-Manα1-Ser-pyridylaminoethylsuccinamyl (PAES). Consistent with the above data, GnT-IX generated a new product which was identified as GlcNAcβ1,2-(GlcNAcβ1,6-)Manα1-Ser-PAES by mass spectrometry and 1H NMR. Furthermore, incorporation of an additional GlcNAc residue into a synthetic mannosyl peptide Ac-Ala-Ala-Pro-Thr(Man)-Pro-Val-Ala-Ala-Pro-NH2 by GnT-IX was only observed in the presence of POMGnT1. Collectively, these results strongly suggest that GnT-IX may be a novel β1,6-N-acetylglucosaminyltransferase that is responsible for the formation of the 2,6-branched structure in the brain O-mannosyl glycan.

Cloning and characterization of a novel UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, pp-GalNAc-T14☆

A novel member of the human UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (pp-GalNAc-T) gene family was cloned and designated pp-GalNAc-T14. This type II membrane protein contains all motifs that are conserved in the pp-GalNAc-T family proteins and forms a subfamily with pp-GalNAc-T2 on the phylogenetic tree. Quantitative real time PCR analysis revealed significantly high expression of the pp-GalNAc-T14 transcript in kidney, although the transcripts were ubiquitously expressed in all tissues examined. Furthermore, the recombinant pp-GalNAc-T14 transferred GalNAc to a panel of mucin-derived peptide substrates such as Muc2, Muc5AC, Muc7, and Muc13 (-58). Our results provide evidence that pp-GalNAc-T14 is a new member of the pp-GalNAc-T family and suggest that pp-GalNAc-T14 may be involved in the O-glycosylation in kidney.

α1,3-Fucosyltransferase 9 (FUT9; Fuc-TIX) preferentially fucosylates the distal GlcNAc residue of polylactosamine chain while the other four α1,3FUT members preferentially fucosylate the inner GlcNAc residue

We analyzed the substrate specificity of six human α1,3‐fucosyltransferases (α1,3FUTs) for the 2‐aminobenzamide (2AB)‐labelled polylactosamine acceptor, Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4GlcNAc‐2AB (3LN‐2AB). FUT9 preferentially fucosylated the distal GlcNAc residue of the polylactosamine chain while the other four α1,3FUT members, FUT3, FUT4, FUT5 and FUT6, preferentially fucosylated the inner GlcNAc residue. This indicated that FUT9 exhibits more efficient activity for the synthesis of Lewis x carbohydrate epitope (Lex; CD15; stage‐specific embryonal antigen‐1 (SSEA‐1)). In contrast, the other four members synthesize more effectively the internal Lex epitope. FUT7 could not transfer a fucose to an acceptor which is non‐sialylated.

Molecular Cloning and Characterization of a Novel Human β1,4-N-Acetylgalactosaminyltransferase, β4GalNAc-T3, Responsible for the Synthesis of N,N′-Diacetyllactosediamine, GalNAcβ1–4GlcNAc

We found a novel human glycosyltransferase gene carrying a hypothetical β1,4-glycosyltransferase motif during a BLAST search, and we cloned its full-length open reading frame by using the 5′-rapid amplification of cDNA ends method. It encodes a type II transmembrane protein of 999 amino acids with homology to chondroitin sulfate synthase in its C-terminal region (GenBank™ accession number AB089940). Its putative orthologous gene was also found in mouse (accession number AB114826). The truncated form of the human enzyme was expressed in HEK293T cells as a soluble protein. The recombinant enzyme transferred GalNAc to GlcNAc β-benzyl. The product was deduced to be GalNAcβ1–4GlcNAcβ-benzyl based on mass spectrometry and NMR spectroscopy. We renamed the enzyme β1,4-N-acetylgalactosaminyltransferase-III (β4GalNAc-T3). β4GalNAc-T3 effectively synthesized N,N′-diacetylgalactosediamine, GalNAcβ1–4GlcNAc, at non-reducing termini of various acceptors derived not only from N-glycans but also from O-glycans. Quantitative real time PCR analysis showed that its transcript was highly expressed in stomach, colon, and testis. As some glycohormones contain N,N′-diacetylgalactosediamine structures in their N-glycans, we examined the ability of β4GalNAc-T3 to synthesize N,N′-diacetylgalactosediamine structures in N-glycans on a model protein. When fetal calf fetuin treated with neuraminidase and β1,4-galactosidase was utilized as an acceptor protein, β4GalNAc-T3 transferred GalNAc to it. Furthermore, the majority of the signal from GalNAc disappeared on treatment with glycopeptidase F. These results suggest that β4GalNAc-T3 could transfer GalNAc residues, producing N,N′-diacetylgalactosediamine structures at least in N-glycans and probably in both N- and O-glycans.

Molecular cloning and characterization of a novel member of the UDP‐GalNAc:polypeptide N‐acetylgalactosaminyltransferase family, pp‐GalNAc‐T121

We cloned in silico a novel human UDP‐GalNAc:polypeptide N‐acetylgalactosaminyltransferase (pp‐GalNAc‐T), pp‐GalNAc‐T12. The deduced amino acid sequence of pp‐GalNAc‐T12 contains all conserved motifs in pp‐GalNAc‐T family proteins. Quantitative real time polymerase chain reaction analysis revealed that the pp‐GalNAc‐T12 transcript was expressed mainly in digestive organs such as stomach, small intestine and colon. The recombinant pp‐GalNAc‐T12 transferred GalNAc to the mucin‐derived peptides such as the Muc1a, Muc5AC, EA2 peptides and the GalNAc‐Muc5AC glycopeptide. Since mucins are glycoproteins mainly produced in the digestive organs, our results suggest that pp‐GalNAc‐T12 plays an important role in the initial step of mucin‐type oligosaccharide biosynthesis in digestive organs.

Up‐regulation of Lewis enzyme (Fuc‐TIII) and plasma‐type α1,3Fucosyltransferase (Fuc‐TVI) expression determines the augmented expression of sialyl Lewis x antigen in non‐small cell lung cancer

Sialyl Lewis a and x antigens are well‐known tumor‐associated antigens expressed in many cancer tissues. The expression of the genes encoding 5 α1,3fucosyltransferases, which are able to synthesize the sialyl Lewis antigens, was examined in normal and cancerous lung tissues of patients with non‐small cell lung carcinoma. In all 20 cases examined, the transcripts only for the Lewis gene, encoding the Lewis enzyme (α1,3/4fucosyltransferase, Fuc‐TIII), were abundantly expressed in lung tissue, and interestingly they were markedly up‐regulated in the lung cancer tissues of all 20 cases in comparison with normal lung tissues. Myeloid‐type α1,3fucosyltransferase (Fuc‐TIV) was expressed at an intermediate level but was not up‐regulated in lung cancer tissues. The transcripts for plasma‐type α1,3fucosyltransferase (Fuc‐TVI) gene were detected at a very low level but were apparently up‐regulated in cancer tissues. Fuc‐TVI was found to exhibit stronger relative activity for sialyl Lewis x synthesis (almost 6.4‐fold that of Fuc‐TIII). The amount of sialyl Lewis x antigen on mucins in the lung cancer tissues was found to be determined by both enzymes, the Lewis enzyme (Fuc‐TIII) and Fuc‐TVI. However, the amount of the sialyl Lewis a antigens was not determined by any of the α1,3‐fucosyltransferases, although the expression of sialyl Lewis a antigens definitely required the Lewis enzyme. Int. J. Cancer 83:70–79, 1999.

Murine monoclonal antibody recognizing human alpha(1,3/1,4)fucosyltransferase.

We prepared a mouse monoclonal antibody, FTA1-16, that specifically recognizes human α(1,3/1,4)fucosyltransferase without crossreactivity to any other members of the α(1,3)fucosyltransferase family. The specificity was confirmed by both immunofluorescense staining of native antigens in the Golgi apparatus and Western blotting analysis, using stable transformant cells transfected with each gene of the α(1,3)fucosyltransferase family. Western blotting analysis on a series of human tumour cell lines from various tissues revealed that some epithelial cancer cell lines from digestive organs expressed an amount of α(1,3/1,4)fucosyltransferase in good correlation with expression of sialyl Lewis a antigen. Immunohistochemical staining by FTA1-16 on colon cancer tissues revealed enhanced expression of the enzyme in cancer cells in comparison to normal cells. Finally, the antigenic epitope recognized by FTA1-16 was determined using truncated recombinant peptides which were expressed inE. coli. A minimal length determined was a fragment, amino acid positions 132–153, of the α(1,3/1,4)fucosyltransferase.