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Yukishige Ito

Publications and source records attributed to Yukishige Ito.

At least 19 recordsLinked to original sources
PubMed2006

Detection of weak sugar binding activity of VIP36 using VIP36-streptavidin complex and membrane-based sugar chains.

High mannose-type glycan-lectin interactions play important roles especially in quality control of glycoproteins. VIP36 is a receptor with homology to plant leguminous lectins in its luminal region. The luminal region of VIP36 with a C-terminal biotinylation-tag (sVIP36) was expressed in Escherichia coli and oligomerized with R-phycoerythrin (PE)-labelled streptavidin. Flow cytometric analysis revealed that PE-labelled sVIP36-SA complex (sVIP36-SA) bound to deoxymannojirimycin (DMJ)- and kifunensine (KIF)-treated HeLaS3 cells. The binding of sVIP36-SA to HeLaS3 cells treated with DMJ or KIF was abolished by endo-beta-N-acetylglucosaminidase H treatment of the cells. Furthermore, the binding of sVIP36-SA to the cells was inhibited by high mannose-type glycans especially Man(7-9) GlcNAc(2), indicating that the binding of sVIP36-SA to cell surfaces was mediated by high mannose-type glycans. Although VIP36 has the lower affinity for ligands than typical homologous plant lectins, we were able to monitor the sugar-binding activity of VIP36 using less than 100 ng of the sVIP36-SA. This method is highly sensitive and suitable for detecting interactions between lectins and sugar chains of low affinity.

1-Deoxynojirimycin↗
PubMed2006

Stereoselective synthesis of a fragment of mycobacterial arabinan.

Strategies for the stereoselective synthesis of mycobacterial arabinan were explored. Arabinofuranosyl donors with various protective groups were screened in terms of suitability for beta-(1,2-cis)-selective glycosylation. The protective group was found to affect the stereoselectivity of arabinofuranosylation. Beta-selectivity was drastically enhanced by using donors protected with 3,5-TIDPS, possibly due to conformational constraints on the furanose ring. Synthesis of heptaarabinofuranoside was then performed to demonstrate the practicality of this methodology. [reaction: see text].

Carbohydrate Sequence↗
PubMed2006

Substrate specificity analysis of endoplasmic reticulum glucosidase II using synthetic high mannose-type glycans.

Glucosidase II (Glc'ase II) is a glycan-processing enzyme that trims two alpha1,3-linked Glc residues in succession from the glycoprotein oligosaccharide Glc2Man9GlcNAc2 to give Glc1Man9GlcNAc2 and Man9GlcNAc2 in the endoplasmic reticulum (ER). Monoglucosylated glycans, such as Glc1-Man9GlcNAc2, generated by this process play a key role in glycoprotein quality control in the ER, because they are primary ligands for the lectin chaperones calnexin (CNX) and calreticulin (CRT). A precise analysis of the substrate specificity of Glc'ase II is expected to further our understanding of the molecular basis to glycoprotein quality control, because Glc'ase II potentially competes with CNX/CRT for the same glycans, Glc1Man7-9GlcNAc2. In this study, a quantitative analysis of the specificity of Glc'ase II using a series of structurally defined synthetic glycans was carried out. In the presence of CRT, Glc'ase II-mediated trimming from Glc2Man9GlcNAc2 stopped at Glc1Man9GlcNAc2, supporting the notion that the glycan structure delivered to the CNX/CRT cycle is Glc1Man9GlcNAc2. Unexpectedly, our experiments showed that Glc1Man8(B)GlcNAc2 had nearly the same reactivity as Glc1Man9GlcNAc2, which was markedly greater than that of its positional isomer Glc1Man8(C)GlcNAc2. An analysis with glycoprotein-like probes revealed the stepwise formation of Glc1Man9GlcNAc2 and Man9GlcNAc2 from Glc2Man9GlcNAc2, even in the presence of CRT. It was also shown that Glc1Man8(B)GlcNAc2 had even greater reactivity than Glc1Man9GlcNAc2 at the glycoprotein level. Moreover, inhibitory activities by nonglucosylated glycans suggested that Glc'ase II recognized the C arm (Manalpha1, 2Manalpha1, 6Man-) of high mannose-type glycans.

Animals↗
PubMed2006

N-benzyl-2,3-oxazolidinone as a glycosyl donor for selective alpha-glycosylation and one-pot oligosaccharide synthesis involving 1,2-cis-glycosylation.

Glycosylation reactions using N-benzyl-2,3-trans-oxazolidinones as the glycosyl donors were shown to be highly alpha-selective. Advantages of the donor include facile preparation in gram-scale preparation and simple deprotection procedures. Subsequently, a one-pot oligosaccharide synthesis involving 1,2-cis glycosidic linkages was demonstrated using the novel glycosyl donors.

Journal Article↗
PubMed2006

Site-specific labeling of cytoplasmic peptide:N-glycanase by N,N'-diacetylchitobiose-related compounds.

Peptide:N-glycanase (PNGase) is the deglycosylating enzyme, which releases N-linked glycan chains from N-linked glycopeptides and glycoproteins. Recent studies have revealed that the cytoplasmic PNGase is involved in the degradation of misfolded/unassembled glycoproteins. This enzyme has a Cys, His, and Asp catalytic triad, which is required for its enzymatic activity and can be inhibited by "free" N-linked glycans. These observations prompted us to investigate the possible use of haloacetamidyl derivatives of N-glycans as potent inhibitors and labeling reagents of this enzyme. Using a cytoplasmic PNGase from budding yeast (Png1), Man9GlcNAc2-iodoacetoamide was shown to be a strong inhibitor of this enzyme. The inhibition was found to be through covalent binding of the carbohydrate to a single Cys residue on Png1, and the binding was highly selective. The mutant enzyme in which Cys191 of the catalytic triad was changed to Ala did not bind to the carbohydrate probe, suggesting that the catalytic Cys is the binding site for this compound. Precise determination of the carbohydrate attachment site by mass spectrometry clearly identified Cys191 as the site of covalent attachment. Molecular modeling of N,N'-diacetylchitobiose (chitobiose) binding to the protein suggests that the carbohydrate binding site is distinct from but adjacent to that of Z-VAD-fmk, a peptide-based inhibitor of this enzyme. These results suggest that cytoplasmic PNGase has a separate binding site for chitobiose and other carbohydrates, and haloacetamide derivatives can irreversibly inhibit that catalytic Cys in a highly specific manner.

Amino Acid Substitution↗
PubMed2006

Synthesis of asparagine-linked bacillosamine.

Various types of protein glycosylation have been identified from prokaryotes. Recent investigations have revealed the presence of N-linked glycoproteins in the pathogenic bacterium, Campylobacter jejuni. The structure of this glycan is unique, consisting of 5 GalNAc and 1 Glc, in addition to 2,4-diacetamido-2,4,6-trideoxy-d-glucopyranose (bacillosamine; Bac), which is N-glycosidically linked to the side chain of asparagine (Asn). We synthesized Bac from a 2-azido-2-deoxy-D-galactose derivative, which was further converted to the Asn-linked form.

Asparagine↗
PubMed2006

High-mannose-type glycan modifications of dihydrofolate reductase using glycan-methotrexate conjugates.

Various high-mannose-type glycan modifications of dihydrofolate reductase (DHFR) were achieved by ligand-based approach using glycan-methotrexate (MTX) conjugates as tight binding glycan bearing ligands for DHFR. The resulting glycan-MTX conjugates and the corresponding artificial glycoproteins could be useful as oligosaccharide- and glycoprotein-probes to perform quantitative analysis of glycan recognizing protein such as lectins, glycosyltransferases or glycosidases. Moreover, artificial glycoproteins having two different high-mannose-type glycans were developed for the first time by a combination of two different types of glycan modification strategies.

Escherichia coli↗
PubMed2006

Systematic synthesis of bisubstrate-type inhibitors of N-acetylglucosaminyltransferases.

Bisubstrate-type inhibitors for N-acetylglucosaminyltransferase (GnT)-V and -IX were designed and synthesized. These compounds carry both an acceptor trisaccaride and an UDP-GlcNAc component tethered by a linker of variable length. The acceptor trisaccharide unit was constructed using a combination of a polymer support and a resin capture-release strategy. Namely, starting with a beta-mannoside bound to low molecular weight monomethyl PEG (MPEG), successive glycosylations with donors having chloroacetyl group produced the trisaccharide, which was subjected to the capture-release purification using cysteine loaded resin. UDP-GlcNAc units carrying phosphate moieties were separately synthesized from the bromoacetamide-containing glucosamine derivative. Ligation between the acceptor thiol and each alkyl bromide on the donor unit readily proceeded, and produced the coupling product. The introduction of the UMP component gave target compounds. All of the synthesized compounds had significant activities to GnT-V and -IX. Their potencies were dependent upon the linkers length. GnT-IX was more sensitive to these inhibitors and optimum linker length was clearly different between these GnTs. The most potent inhibitor of GnT-V had Ki=18.3 microM, while that of GnT-IX had Ki = 4.7 microM.

Carbohydrate Sequence↗
PubMed2006

A stereoselective 1,2-cis glycosylation toward the synthesis of a novel N-linked glycan from the Gram-negative bacterium, Campylobacter jejuni.

It has been shown that certain prokaryotes, such as Campylobacter jejuni, have asparagine (Asn)-linked glycoproteins. However, the structures of their glycans are distinct from those of eukaryotic origin. They consist of a bacillosamine residue linked to Asn, an alpha-(1-->4)-GalpNAc repeat, and a branching beta-Glcp residue. In this paper, we describe a strategy for the stereoselective construction of the alpha-(1-->4)-GalpNAc repeat of a C. jejuni N-glycan, utilizing a pentafluoropropionyl (PFP) group as a temporary protective group of the C-4 OH group of the GalpN donor. The strategy was applied to the synthesis of the hexasaccharide alpha-GalpNAc-(1-->4)-alpha-GalpNAc-(1-->4)-[beta-Glcp-(1-->3)]-alpha-GalpNAc(1-->4)-alpha-GalpNAc-(1-->4)-GalpNAc.

Acetylgalactosamine↗
PubMed2006

Structural characterization of glycopeptides by N-terminal protein ladder sequencing.

High-sensitivity and high-throughput mass spectrometry (MS) has become an important tool for characterizing glycopeptides. Here, we analyzed synthetic O-linked glycopeptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. First, we applied MALDI-quadrupole ion trap (QIT)-TOF MS, which enables collision-induced dissociation-MSn analysis for fine structural characterization. Subsequent MS/MS of sodium adduct ions selected as precursor ions yielded detailed information about the site of oligosaccharide attachment as well as the carbohydrate and amino acid sequences; however, these MS/MS spectra were very complex. To obtain easily interpretable and simple spectra, we used N-terminal protein ladder sequencing coupled with MALDI-TOF MS. From the extremely simple resulting spectra, we were able to determine the glycosylation sites, amino acid sequences, and oligosaccharide molecular weights of the glycopeptides.

Amino Acid Sequence↗
PubMed2006

Synthesis and TNF-alpha inducing activities of mycoloyl-arabinan motif of mycobacterial cell wall components.

The extract of the cell wall skeleton of Bacillus Calmette-Guérin (BCG-CWS) from Mycobacterium bovis is known to be an activator of innate immunity. Synthesis of pentaarabinofuranoside as part of the arabinan moiety of BCG-CWS was achieved by double alpha-arabinofuranosylation followed by double beta-arabinofuranosylation with orthogonally protected donors. Mycolic esters of the arabinan in the terminal lipo-arabinan motif of BCG-CWS were synthesized through alkylation of unprotected mycolic acid with bis- and tetra-tosylates of pentaarabinofuranoside. A series of compounds were subjected to a tumor necrosis factor alpha (TNF-alpha) secretion-inducing assay, disclosing aspects of the structure-activity relationship which should be useful in finding the site of the activity.

Animals↗
PubMed2005

Synthesis of complex-type glycans derived from parasitic helminths.

Chemical synthesis of complex-type glycans 1 and 2 derived from eggs of parasitic helminths, Schistosoma mansoni and Schistosoma japonicum, is described. These branched sugar chains were synthesized regio- and stereoselectively by using beta-mannosylation, desilylation under high pressure, and glycosylation in frozen solvent as key transformations.

Animals↗
PubMed2005

Sugar-binding properties of VIP36, an intracellular animal lectin operating as a cargo receptor.

The vesicular integral protein of 36 kDa (VIP36) is an intracellular animal lectin that acts as a putative cargo receptor, which recycles between the Golgi and the endoplasmic reticulum. Although it is known that VIP36 interacts with glycoproteins carrying high mannose-type oligosaccharides, detailed analyses of the sugar-binding specificity that discriminates isomeric oligosaccharide structures have not yet been performed. In the present study, we have analyzed, using the frontal affinity chromatography (FAC) method, the sugar-binding properties of a recombinant carbohydrate recognition domain of VIP36 (VIP36-CRD). For this purpose, a pyridylaminated sugar library, consisting of 21 kinds of oligosaccharides, including isomeric structures, was prepared and subjected to FAC analyses. The FAC data have shown that glucosylation and trimming of the D1 mannosyl branch interfere with the binding of VIP36-CRD. VIP36-CRD exhibits a bell-shaped pH dependence of sugar binding with an optimal pH value of approximately 6.5. By inspection of the specificity and optimal pH value of the sugar binding of VIP36 and its subcellular localization, together with the organellar pH, we suggest that VIP36 binds glycoproteins that retain the intact D1 mannosyl branch in the cis-Golgi network and recycles to the endoplasmic reticulum where, due to higher pH, it releases its cargos, thereby contributing to the quality control of glycoproteins.

Chromatography, Affinity↗
PubMed2005

Thermodynamic analysis of interactions between N-linked sugar chains and F-box protein Fbs1.

Fbs1 is a recently discovered F-box protein that was proposed to recognize high-mannose-type asparagine-linked glycoprotein sugar chains. To reveal the specificity of Fbs1, Manalpha1-->6Manbeta1-->4GlcNAc(2), Manalpha1-->3Manbeta1-->4GlcNAc(2), and Manalpha1-->3(Manalpha1--> 6)Manbeta1-->4GlcNAc(2) were synthesized and their affinities for Fbs1 were evaluated in comparison with previously synthesized Man(9)GlcNAc(2) and Man(8)GlcNAc(2). These analyses revealed that Man(3)GlcNAc(2) had the strongest affinity and the chitobiose and alpha1-->6 linked Man residue are necessary for Fbs1 to recognize a sugar.

Carbohydrate Sequence↗
PubMed2005

Design and synthesis of oligosaccharides that interfere with glycoprotein quality-control systems.

Calnexin (CNX) and its soluble homologue calreticulin (CRT) are lectin-like molecular chaperones that help newly synthesized glycoproteins to fold correctly in the rough endoplasmic reticulum (ER). To investigate the mechanism of glycoprotein-quality control, we have synthesized structurally defined high-mannose-type oligosaccharides related to this system. This paper describes the synthesis of the non-natural undecasaccharide 2 and heptasaccharide 16, designed as potential inhibitors of the ER quality-control system. Each possesses the key tetrasaccharide element (Glc1Man3) critical for the CNX/CRT binding, while lacking the pentamannosyl branch required for glucosidase II recognition. These oligosaccharides were evaluated for their ability to bind CRT by isothermal titration calorimetry (ITC). As expected, each of them had a significant affinity towards CRT. In addition, these compounds were shown to be resistant to glucosidase II digestion. Their activities in blocking the chaperone function of CRT were next measured by using malate dehydrogenase (MDH) as a substrate. Their inhibitory effects were shown to correlate well with their CRT-binding affinities, both being critically dependent upon the presence of the terminal glucose (Glc) residue.

Calnexin↗
PubMed2005

Structural approaches to the study of oligosaccharides in glycoprotein quality control.

High-mannose-type oligosaccharides have been shown to play important roles in protein quality control. Several intracellular proteins, such as lectins, chaperones and glycan-processing enzymes, are involved in this process. These include calnexin/calreticulin, UDP-glucose:glycoprotein glucosyltransferase (UGGT), cargo receptors (such as VIP36 and ERGIC-53), mannosidase-like proteins (e.g. EDEM and Htm1p) and ubiquitin ligase (Fbs). They are thought to recognize high-mannose-type glycans with subtly different structures, although the precise specificities are yet to be clarified. In order to gain a clear understanding of these protein-carbohydrate interactions, comprehensive synthesis of high-mannose-type glycans was conducted. In addition, two approaches to the synthesis of artificial glycoproteins with homogeneous oligosaccharides were investigated. Furthermore, a novel substrate of UGGT was discovered.

Animals↗