Hisashi Narimatsu

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.

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.

A novel glycosyltransferase with a polyglutamine repeat; a new candidate for GD1α synthase (ST6GalNAc V)1

The fifth type GalNAcα2,6‐sialyltransferase (mST6GalNAc V) was cloned from a mouse brain cDNA library. mST6GalNAc V exhibited type II transmembrane topology containing a polyglutamine repeat, which showed 42.6% and 44.8% identity to mouse ST6GalNAc III and IV, respectively. Northern blot analysis revealed that the mST6GalNAc V gene was specifically expressed in forebrain and cerebellum. mST6GalNAc V exhibited GD1α synthetic activity from GM1b the same as mST6GalNAc III and IV. The activity ratio of GM1b toward fetuin and the expression pattern were completely different among the three ST6GalNAcs. Interestingly, the polyglutamine repeat number was different from that of inbred mice. We report the first glycosyltransferase with a polymorphic polyglutamine repeat.

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.

HLA class II antigens are associated with Japanese pemphigus patients

We investigated the HLA class II antigens in 30 Japanese cases of pemphigus, 17 cases of pemphigus vulgaris (PV) and 13 cases of pemphigus foliaceus (PF), by both serologic and restriction fragment length polymorphism (RFLP) analyses. We detected two major haplotypes susceptible to PV, i.e., DRw12-DQw7 and DRw6-DQw5. In contrast, DR2 was absent in PV. RFLP analyses showed that DRw6 PV patients had a disease-associated restriction fragment representing DQw5, the same association as that found in DRw6 Jewish PV patients. However, DRw12 Japanese PV patients had DQw7, whereas DR4 Jewish PV patients had DQw8. On the other hand, all 13 PF patients were serologically typed for DQw1, which could not be further subdivided into DQw5 by RFLP analyses. These results suggest that Japanese and Jewish PV patients may be immunogenetically closely related to each other, but Japanese PV patients appear to be immunogenetically different from Japanese PF patients.

Recent Progress in Molecular Cloning of Glycosyltransferase Genes of Eukaryotes

The enormous diversity of carbohydrate structures of glycoconjugates is biosynthesized in order by a series of glycosyltransferases. These enzymes have strict substrate specificities. No transferase can utilize more than one type of sugar donor. They can clearly recognize even subtle differences among acceptor molecules and different intersugar-linkages. Hence, it is considered that there are numerous different glycosyltransferases. They are classified into families according to what kind of donor substrates is utilized for sugar transfer. The nine kinds of sugar nucleotides as donor substrates for eukaryote enzymes are listed in Table 1. The family of the enzymes which tansfer the galactose from UDP-galactose (UDP-Gal) to the non-reducing terminus of the sugar acceptor substrate is called galactosyltransferases (GalTs). A UDP-Gal: N-acetylglucosamine ƒÀ1, 4 galactosyltransferase (ƒÀ1,4 Ga1T), a member of the GalTs, catalyzes the transfer of galactose from UDP-Gal to the nonreducing end of N-acetyl-

Synthesis and characterization of a carbene-generating biotinylated N-acetylglucosamine for photoaffinity labeling of β-(1 → 4)-galactosyltransferase

A photoreactive N-acetylglucosamine derivative, N-[2-[2-[2-(2-biotinylaminoethoxy)-ethoxy]ethoxy]-4-[3-(trifluo rom ethyl)-3-H-diazirin-3-yl]benzoyl]-N4-[2-(acetylamino)-deoxy-beta-D -glucopyranosyl]-L-aspartamide (BDGA), was synthesized as a carbene-generating biotinylated probe for UDP-galactose:N-acetylglucosamine beta-(1-->4)-galactosyltransferase (GalT). The photoaffinity labeling experiments of bovine GalT with BDGA under various condition were examined based on the quantitative chemiluminescent detection of the biotinyl residue which was photochemically introduced into in GalT protein. A progressive decrease in the yield of specific photolabeling was observed upon lowering the incubation temperature from 37 degrees C to 20 degrees C or 4 degree C. The amount of photoincorporation was also decreased when UMP was not included in the incubation mixture. Using a crude protein mixture of recombinant human GalT, a band corresponding to the glutathione S-transferase fusion GalT protein was also specifically visualized. Furthermore, combine use of BDGA photolabeling with an immobilized avidin was found to be effective for the selective retrieval of photolabeled GalT from a reaction mixture containing a large amount of unlabeled GalT protein. The results obtained clearly demonstrate that the covalent biotinylation using the carbene-generating photoaffinity reagent BDGA would be useful for the analysis of acceptor substrate binding sites within the GalT protein.

Assignment1 of the human α 1,3-fucosyltransferase IX gene (FUT9) to chromosome band 6q16 by in situ hybridization

Lewis x (Lex) is defined as the terminal structure of carbohydrate chains consisting of the trisaccharide structure, Galß14(Fuc·1-3)GlcNAc, and is considered to play important roles in cell-cell interactions during embryonic development, differentiation, and oncogenesis. We recently isolated a novel human ·1,3-fucosyltransferase gene (FUT9, alias hFucTIX), which is involved in the last step of Lex synthesis (Kaneko et al., 1999). The amino acid sequences of FUT9 are very highly conserved between human and the mouse homologue (Kudo et al., 1998; Kaneko et al., 1999). This high conservation is not the case for other fucosyltransferases which were previously cloned. FUT9 has been under a strong selective pressure during its evolution, suggesting that it plays essential roles in ontogeny. We report here the chromosomal mapping of FUT9. Materials and methods

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.

Association between Expression Levels of CA 19-9 and N-Acetylglucosamine-β1,3-Galactosyltransferase 5 Gene in Human Pancreatic Cancer Tissue

Objective: CA 19-9, equivalent to Sialyl Lewisa antigen, is a well-known tumor marker in pancreatic cancer. At the initial step of the biosynthesis of CA 19-9, N-acetylglucosamine-β1,3-galactosyltransferase (β3Gal-T) transfers galactose to N-acetylglucosamine (GlcNAc). Recently, β3Gal-T5 has been presumed to be related to the formation of the type 1 chain in an in vitro experiment in terms of kinetic enzyme characterization. The purpose of this study was to investigate which β3Gal-T is related to the synthesis of CA 19-9 in human pancreatic cancer tissues. Methods: We examined β3Gal-T1, T2, T3, T4, and β3Gal-T5 mRNA expressions in 13 noncancerous and cancerous tissues of the human pancreas using real-time polymerase chain reaction, and compared those gene expression levels with the immunoreactivity of CA 19-9 and its precursor DUPAN-2 in cancerous tissues. Results:β3Gal-T5 gene expression significantly augmented in cancerous tissues, when compared with the adjacent noncancerous tissues. Additionally, there was a good correlation between β3Gal-T5 gene transcription levels and immunohistochemical grades of CA 19-9 or its precursor DUPAN-2 in cancerous tissues. However, no correlation was observed between β3Gal-T1, T2, T3, and β3Gal-T4 gene expression levels and CA 19-9 or DUPAN-2 immunoreactive grades in cancerous tissue. Conclusion: β3Gal-T5 is presumed to be responsible for the synthesis of CA 19-9 in pancreatic cancer tissue.