Takao Ohashi

Critical Roles of Mucin 1 Glycosylation by Transactivated Polypeptide N-Acetylgalactosaminyltransferase 6 in Mammary Carcinogenesis

The structure of O-glycosylated proteins is altered in breast cancer cells, but the mechanisms of such an aberrant modification have been largely unknown. We here report critical roles of a novel druggable target, polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), which is upregulated in a great majority of breast cancers and encodes a glycosyltransferase responsible for initiating mucin-type O-glycosylation. Knockdown of GALNT6 by small interfering RNA significantly enhanced cell adhesion function and suppressed the growth of breast cancer cells. Western blot and immunostaining analyses indicated that wild-type GALNT6 protein could glycosylate and stabilize an oncoprotein mucin 1 (MUC1), which was upregulated with GALNT6 in breast cancer specimens. Furthermore, knockdown of GALNT6 or MUC1 led to similar morphologic changes of cancer cells accompanied by the increase of cell adhesion molecules beta-catenin and E-cadherin. Our findings implied that overexpression of GALNT6 might contribute to mammary carcinogenesis through aberrant glycosylation and stabilization of MUC1 and that screening of GALNT6 inhibitors would be valuable for the development of novel therapeutic modalities against breast cancer.

Production of heterologous proteins using the fission-yeast (Schizosaccharomyces pombe) expression system

The fission yeast Schizosaccharomyces pombe is a particularly useful model for studying the function and regulation of genes from higher eukaryotes. The genome of Sc. pombe has been sequenced, and DNA microarray, proteome and transcriptome analyses have been carried out. Among the well‐characterized yeast species, Sc. pombe is considered an attractive host for the production of heterologous proteins. Expression vectors for high‐level expression in Sc. pombe have been developed and many foreign proteins have been successfully expressed. However, further improvements in the protein‐expressing host systems are still required for the production of heterologous proteins involved in post‐translational modification, metabolism and intracellular trafficking. This minireview focuses on recent advances in heterologous protein production by use of engineered fission‐yeast strains.

Identification and characterization of a gene required for α1,2-mannose extension in the O-linked glycan synthesis pathway in Schizosaccharomyces pombe

The KTRalpha1,2-mannosyltransferase gene family of Saccharomyces cerevisiae is responsible not only for outer-chain modifications of N-linked oligosaccharides but also for elongation of O-linked mannose residues. To identify genes involved in the elongation step of O-linked oligosaccharide chains in Schizosaccharomyces pombe, we characterized six genes, omh1(+)-omh6(+), that share significant sequence similarity to the S. cerevisiae KTR family. Six deletion strains were constructed, each carrying a single disrupted omh allele. All strains were viable, indicating that none of the omh genes was essential. Heterologous expression of a chitinase from S. cerevisiae in the omh mutants revealed that O-glycosylation of chitinase had decreased in omh1Delta cells, but not in the other mutants, indicating that the other omh genes do not appear to be required for O-glycan synthesis. Addition of the second alpha1,2-linked mannose residue was blocked in omh1Delta cells. An Omh1-GFP fusion protein was found to be localized in the Golgi apparatus. These results indicate that Omh1p plays a major role in extending alpha1,2-linked mannose in the O-glycan pathway in S. pombe.

Characterization of oleaginous yeasts accumulating high levels of lipid when cultivated in glycerol and their potential for lipid production from biodiesel-derived crude glycerol

This study attempted to identify oleaginous yeasts and selected the strain that accumulated the largest quantity of lipid for lipid production from glycerol. Two-step screening of 387 yeast strains revealed 23 oleaginous strains that accumulated quantities of lipid higher than 20 % of their biomass when cultivated in glycerol. These strains were identified to be four ascomycetous yeast species i.e. Candida silvae, Kodamaea ohmeri, Meyerozyma caribbica, and Pichia manshurica, and five basidiomycetous yeast species i.e. Cryptococcus cf. podzolicus, Cryptococcus laurentii, Rhodosporidium fluviale, Rhodotorula taiwanensis, and Sporidiobolus ruineniae. Rhodosporidium fluviale DMKU-RK253 accumulated the highest quantity of lipid equal to 65.2 % of its biomass (3.9 g L(-1) lipid and 6.0 g L(-1) biomass) by shaking flask cultivation in crude glycerol. The main fatty acids in the accumulated lipid of this strain consisted of oleic acid, linoleic acid, and palmitic acid. Therefore, R. fluviale DMKU-RK253 has potential for producing lipid for biodiesel manufacturing using crude glycerol as a feedstock.

N- and O-linked oligosaccharides completely lack galactose residues in the gms1och1 mutant of Schizosaccharomyces pombe

Unlike their counterparts in budding yeast Saccharomyces cerevisiae, the glycoproteins of Schizosaccharomyces pombe contain, in addition to α-d-mannose (Man), a large number of α-d-galactose (Gal) residues. In both yeasts, large outer chains are attached to the oligosaccharide cores of glycoproteins during export via Golgi. Formation of the yeast-specific large outer chain is initiated by α-1,6-mannosylatransferase encoded by the och1+ gene, the disruption of which blocked outer chain elongation. We previously reported that N-linked oligosaccharide structures of S. pombe och1Δ mutant consisted of Gal2–6Man9GlcNAc2 with α-linked Gal residues attached to the core oligosaccharide moiety. The disruption of gms1+, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, abolished cell surface galactosylation in S. pombe. In this study, we constructed a gms1Δoch1Δ double mutant and determined the N- and O-linked oligosaccharide structures present on the cell surface. Oligosaccharides were liberated from glycoproteins by hydrazinolysis and labeled with the fluorophore, 2-aminopyridine. The pyridylaminated N-linked oligosaccharides were analyzed by high-performance liquid chromatography in combination with α1,2-mannosidase digestion and partial acetolysis. These analyses revealed that the N-linked oligosaccharides of gms1Δoch1Δ cells consisted of α1,2-linked Man-extended core oligosaccharides (Man8–12GlcNAc2) from which the fission yeast-specific α-linked Gal residues were completely absent.

The och1 Mutant of Schizosaccharomyces pombe Produces Galactosylated Core Structures of N-Linked Oligosaccharides

Unlike the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe synthesizes large outer chains on the N-linked oligosaccharides that consist mainly of D-Gal and D-Man residues. The fission yeast och1 + gene product has α1,6-mannosyltransferase activity, and Och1p is the key enzyme in the initiation of outer chain elongation. Although the in vitro substrate specificity of S. pombe Och1p has been reported (Yoko-o et al., FEBS Lett., 489, 75–80 (2001)), the structure of the N-linked oligosaccharides of och1Δ cells has not been investigated. In this study, we report a structural analysis of S. pombe N-linked oligosaccharides. Lectin blot analysis indicated that galactose residues were attached to the cell surface glycoproteins of the och1Δ cells. We conducted a structural analysis of pyridylaminated N-linked oligosaccharides prepared from galactomannoproteins by HPLC and 1H NMR. These analyses revealed that the N-linked oligosaccharides of the och1Δ cells displayed heterogeneity in the glycan consisting of Hex11–15GlcNAc2. The structural heterogeneity arose mainly from the addition of α1,2- and α1,3-Gal residues to the Man9GlcNAc2 core structure.

The production of human glucocerebrosidase in glyco-engineered Nicotiana benthamiana plants.

Summary For the production of therapeutic proteins in plants, the presence of β1,2‐xylose and core α1,3‐fucose on plants’ N‐glycan structures has been debated for their antigenic activity. In this study, RNA interference (RNAi) technology was used to down‐regulate the endogenous N‐acetylglucosaminyltransferase I (GNTI) expression in Nicotiana benthamiana. One glyco‐engineered line (Nb GNTI‐RNAi) showed a strong reduction of plant‐specific N‐glycans, with the result that as much as 90.9% of the total N‐glycans were of high‐mannose type. Therefore, this Nb GNTI‐RNAi would be a promising system for the production of therapeutic glycoproteins in plants. The Nb GNTI‐RNAi plant was cross‐pollinated with transgenic N. benthamiana expressing human glucocerebrosidase (GC). The recombinant GC, which has been used for enzyme replacement therapy in patients with Gauchers disease, requires terminal mannose for its therapeutic efficacy. The N‐glycan structures that were presented on all of the four occupied N‐glycosylation sites of recombinant GC in Nb GNTI‐RNAi plants (GC gnt1) showed that the majority (ranging from 73.3% up to 85.5%) of the N‐glycans had mannose‐type structures lacking potential immunogenic β1,2‐xylose and α1,3‐fucose epitopes. Moreover, GC gnt1 could be taken up into the macrophage cells via mannose receptors, and distributed and taken up into the liver and spleen, the target organs in the treatment of Gauchers disease. Notably, the Nb GNTI‐RNAi line, producing GC, was stable and the Nb GNTI‐RNAi plants were viable and did not show any obvious phenotype. Therefore, it would provide a robust tool for the production of GC with customized N‐glycan structures.

Production of heterologous glycoproteins by a glycosylation-defective alg3och1 mutant of Schizosaccharomyces pombe.

The early stages of N-linked glycosylation are highly conserved between fungal and mammalian cells. Such N-linked oligosaccharides are synthesized through the ordered assembly of a dolichyl pyrophosphate (Dol-PP)-linked Glc(3)Man(9)GlcNAc(2) structure by the sequential actions of several glycosyltransferases located in the endoplasmic reticulum (ER). Of the glycosyltransferase genes, Saccharomyces cerevisiae ALG3 has been identified to encode the Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol α1,3-mannosyltransferase, and an alg3 mutant has been shown to accumulate an Endo H-resistant M5B (Manα1,2-Manα1,2-Manα1,3(Manα1,6-)-Manβ1,4-GlcNAcβ1,4-GlcNAc) structure. Although Schizosaccharomyces pombe contains a homolog of the ALG3 gene (SPAC7D4.06c), the role of this gene in oligosaccharide biosynthesis is not at all clear. In this study, we deleted the alg3(+) gene in the och1Δ mutant and analyzed the detailed oligosaccharide structures in alg3Δoch1Δ double mutant. The oligosaccharides were prepared from cell-surface glycoproteins by hydrazinolysis and fluorescent labeling with 2-aminopyridine. The labeled oligosaccharides were analyzed by high performance liquid chromatography, in combination with sequential glycosidase digestion and methylation analysis. These analyses revealed that the N-linked oligosaccharides of S. pombe alg3Δoch1Δ cells mainly consisted of two or three α-galactose-capped M5B structures. Finally, western blot analysis of recombinant human transferrin suggested that heterologously expressed glycoproteins in alg3Δoch1Δ cells have Endo H-resistant N-linked oligosaccharide structures similar to those of alg3Δoch1Δ cell-surface glycoproteins.

Substrate preference of citrus naringenin rhamnosyltransferases and their application to flavonoid glycoside production in fission yeast

Flavonoids, which comprise a large family of secondary plant metabolites, have received increased attention in recent years due to their wide range of features beneficial to human health. One of the most abundant flavonoid skeletons in citrus species is the flavanone naringenin, which is accumulated as glycosides containing terminal rhamnose (Rha) after serial glycosylation steps. The linkage type of Rha residues is a determining factor in the bitterness of the citrus fruit. Such Rha residues are attached by either an α1,2- or an α1,6-rhamnosyltransferase (1,2RhaT or 1,6RhaT). Although the genes encoding these RhaTs from pummelo (Citrus maxima) and orange (Citrus sinensis) have been functionally characterized, the details of the biochemical characterization, including the substrate preference, remain elusive due to the lack of availability of the UDP-Rha required as substrate. In this study, an efficient UDP-Rha in vivo production system using the engineered fission yeast expressing Arabidopsis thaliana rhamnose synthase 2 (AtRHM2) gene was constructed. The in vitro RhaT assay using the constructed UDP-Rha revealed that recombinant RhaT proteins (Cm1,2RhaT; Cs1,6RhaT; or Cm1,6RhaT), which were heterologously produced in fission yeast, catalyzed the rhamnosyl transfer to naringenin-7-O-glucoside as an acceptor. The substrate preference analysis showed that Cm1,2RhaT had glycosyl transfer activity toward UDP-xylose as well as UDP-Rha. On the other hand, Cs1,6RhaT and Cm1,6RhaT showed rhamnosyltransfer activity toward quercetin-3-O-glucoside in addition to naringenin-7-O-glucoside, indicating weak specificity toward acceptor substrates. Finally, naringin and narirutin from naringenin-7-O-glucoside were produced using the engineered fission yeast expressing the AtRHM2 and the Cm1,2RhaT or the Cs1,6RhaT genes as a whole-cell-biocatalyst.

The fission yeast Pvg1p has galactose-specific pyruvyltransferase activity

N‐Glycan from the fission yeast Schizosaccharomyces pombe contains outer‐chain pyruvic acid 4,6‐ketal‐linked galactose (PvGal). Here, we characterized a putative S. pombe pyruvyltransferase, Pvg1p, reported to be essential for biosynthesis of PvGal. When p‐nitrophenyl‐β‐Gal (pNP‐β‐Gal) was used as a substrate, the structure of the recombinant Pvg1p product was determined to be pNP‐PvGal by one‐ and two‐dimensional NMR spectroscopy. The recombinant Pvg1p transferred pyruvyl residues from phosphoenolpyruvate specifically to β‐linked galactose.