Yukishige Ito

Osaka University | Handai · Graduate School of Science

The cell walls of pathogenic and acidophilic bacteria, such as Mycobacterium tuberculosis and Mycobacterium leprae, contain lipoarabinomannan and arabinogalactan. These components are composed of d-arabinose, the enantiomer of the typical l-arabinose found in plants. The unique glycan structures of mycobacteria contribute to their ability to evade...

Controlling the stereoselectivity of 1,2-cis glycosylation is one of the most challenging tasks in the chemical synthesis of glycans. There are various 1,2-cis glycosides in nature, such as α-glucoside and β-mannoside in glycoproteins, glycolipids, proteoglycans, microbial polysaccharides, and bioactive natural products. In the structure of polysac...

d -Arabinofuranosidases (Ara f ases) degrade the arabinan in the cell wall of acid-fast bacteria like Mycobacterium. Synthetic arabinan fragment probes could be used to investigate the function of d -Ara f ases, whose synthetic studies had been reported previously. The homologue of one of the arabinan-degrading enzymes, exo-α- d -Ara f ase, was ide...

Cell walls of pathogenic and acidophilic bacteria, such as Mycobacterium tuberculosis and Mycobacterium leprae , comprise lipoarabinomannan and arabinogalactan, which are composed of D-arabinose, the enantiomer of the typical l-arabinose found in plants. Their unusual glycan structures serve to immune-evasive of pathogenic mycobacteria. In this stu...

UDP-Glc:glycoprotein glucosyltransferase (UGGT) has a central role to retain quality control of correctly folded N-glycoprotein in the endoplasmic reticulum (ER). A selective and potent inhibitor against UGGT could lead to elucidation of UGGT-related events, but such a molecule has not been identified so far. Examples of small molecules with UGGT i...

In plant cell walls, the hydroxyproline-rich glycoproteins (HRGPs) such as extensin contain oligoarabinofuranoside linked to a hydroxyproline (Hyp) residue. The mature arabinooligosaccharide was revealed to be a tetrasaccharide (α-L-Araf-(1->3)-β-L-Araf-(1->2)-β-L-Araf-(1->2)-β-L-Araf, L-Araf4), whose linkages are targets of the bifidobacterial and...

Understanding the SelenoF‐binding domain of UDP‐glucose:glycoprotein glucosyltransferase (UGGT) and its relative position will provide structural insights into the function of SelenoF. A series of pulldown assays revealed that SelenoF binds to the flexible TRXL2 region of UGGT1. SelenoF forms covalent bonds to a wide range of sites on UGGT1, sugges...

Professor Hidetoshi Yamada, who demonstrated his creativity in various respects, passed away in November 2019. His research targets were highly diverse, including sweet saponins, ellagitannins, novel cyclodextrins, and conformationally distorted donors for stereoselective glycosylations. In memory of his creativity, this chapter highlights his prom...

In the endoplasmic reticulum glycoprotein quality control system, UDP-glucose:glycoprotein glucosyltransferase (UGGT) functions as a folding sensor. Although it is known to form a heterodimer with selenoprotein F (SelenoF), the details of the complex formation remain obscure. A pulldown assay using co-transfected SelenoF and truncated mutants of hu...

To understand the precise mechanism of the glycoside hydrolase (GH) family 127, a cysteine β -L-arabinofuranosidase (Arafase) – HypBA1 – has been isolated from Bifidobacterium longum in the human Gut microbiota, and the design and synthesis of the mechanism-based inhibitors such as L-Araf-haloacetamides have been carried out. The α -L-Araf-azide de...

α1,2‑mannosidase-like proteins mediate quality control of glycoproteins in the endoplasmic reticulum. This study explored α1,2‑mannosidase-like protein functions in Saccharomyces cerevisiae. Single disruptants in targeted protein-coding genes were found to be viable; however, deletion of MNL2 resulted in declined yeast growth at 37 °C. The normal g...

For the stereoselective assembly of bioactive glycans with various functions, 1,2-cis-O-glycosylation is one of the most essential issues in synthetic carbohydrate chemistry. The cis-configured O-glycosidic linkages to the substituents at two positions of the non-reducing side residue of the glycosides such as α-glucopyranoside, α-galactopyranoside...

Pradimicin A (PRM-A) is an actinomycete-derived antibiotic that shows antifungal activity through binding to d-mannose (Man)-containing glycans of fungi. Despite its unique mode of antifungal action, therapeutic application of PRM-A has been severely limited by the aggregate-forming property. In this study, we demonstrated that C4′-N-formyl PRM-A a...

Under ER stress conditions, the ER form of transmembrane proteins can reach the plasma membrane via a Golgi‐independent unconventional protein secretion (UPS) pathway. However, the targeting mechanisms of membrane proteins for UPS are unknown. Here, this study reports that TMED proteins play a critical role in the ER stress‐associated UPS of transm...

Glycosylation is one of the major forms of protein post-translational modification. N -glycans attached to proteins by covalent bonds play an indispensable role in intercellular interaction and immune function. In human bodies, most of the cell surface glycoproteins and secreted glycopeptides are modified with complex-type N -glycans. Thus, for ana...

Methyl β-l-arabinofuranosyl-(1→2)-, -(1→3)-, and -(1→5)-α-l-arabinofuranosides have been stereoselectively synthesized through 2-naphthylmethyl ether-mediated intramolecular aglycon delivery (NAP-IAD), whose β-linkages were confirmed by NMR analysis on the ³JH1–H2 coupling constant and ¹³C chemical shift of C1. The NAP-IAD approach was simply exten...

Oligomannose-type glycans on glycoproteins play an important role in the endoplasmic reticulum (ER)-protein quality control. Mannose trimming of the glycans triggers the ER-associated protein degradation pathway. In mammals, ER mannosyl-oligosaccharide 1,2-α-mannosidase 1 and three ER degradation -enhancing α-mannosidase-like proteins (EDEMs) are r...

Gum arabic is an arabinogalactan protein (AGP) that is effective as a prebiotic for the growth of bifidobacteria in the human intestine. We recently identified a key enzyme in the glycoside hydrolase (GH) family 39, 3- O -α- d -galactosyl-α- l -arabinofuranosidase (GAfase), for the assimilation of gum arabic AGP in Bifidobacterium longum subsp. lon...

Pradimicins (PRMs) are an exceptional family of natural products that specifically bind d-mannose (Man). In the past decade, their scientific significance has increased greatly, with the emergence of biological roles of Man-containing glycans. However, research into the use of PRMs has been severely limited by their inherent tendency to form water-...

s Stereoselective O-glycosylation is essential in order for achieving facile assembly of biologically relevant oligosaccharides. Numerous studies have been conducted to develop glycosylation reactions, whose efficiency and selectivity are influenced by a variety of factors including leaving groups, activators, protective groups, and reaction condit...

Pradimicin A (PRM-A) and its derivatives comprise a unique family of antibiotics that show antifungal, antiviral, and antiparasitic activities through binding to D-mannose (Man)-containing glycans of pathogenic species. Despite their great potential as drug leads with an exceptional antipathogenic action, therapeutic application of PRMs has been se...

C-Mannosylation is a rare type of protein glycosylation and is reportedly critical for the proper folding and secretion of parental proteins. Still, the effects of C-mannosylation on the biological functions of these modified proteins remain to be elucidated. The Trp-x-x-Trp (WxxW) sequences, whose first tryptophan (Trp) can be C-mannosylated, cons...

β-l-Arabinofuranosidase HypBA1 from Bifidobacterium longum belongs to the glycoside hydrolase family 127. At the active site of HypBA1, a cysteine residue (Cys417) coordinates with a Zn2+ atom and functions as the catalytic nucleophile for the anomer-retaining hydrolytic reaction. In this study, the role of Zn2+ ion and cysteine in catalysis as wel...

The Trp-x-x-Trp (W-x-x-W) peptide motif, a consensus site for C-mannosylation, is the functional motif in cytokine type I receptors or thrombospondin type I repeat (TSR) superfamily proteins. W-x-x-W motifs are important for physiological and pathological functions of their parental proteins, but effects of C-mannosylation on protein functions rema...

Fructooligosaccharides and their anhydrides are widely utilized as health-promoting foods and prebiotics. Various enzymes acting on β-D-fructofuranosyl linkages of natural fructan polymers have been utilized to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identi...

Pradimicin A (PRM-A) and related compounds constitute an exceptional family of natural pigments that show Ca2+-dependent recognition of d-mannose (Man). Although these compounds hold great promise as research tools in glycobiology, their practical application has been severely limited by their inherent tendency to form water-insoluble aggregates. H...

C-Mannosylation is a post-translational modification of proteins in the endoplasmic reticulum. Monomeric α-mannose is attached to specific Trp residues at the first Trp in the Trp-x-x-Trp/Cys (W-x-x-W/C) motif of substrate proteins, by the action of C-mannosyltransferases, DPY19-related gene products. The acceptor substrate proteins are included in...

Gum arabic arabinogalactan (AG) protein (AGP) is a unique dietary fiber that is degraded and assimilated by only specific strains of Bifidobacterium longum subsp. longum . Here, we identified a novel 3- O -α- d -galactosyl-α- l -arabinofuranosidase (GAfase) from B. longum JCM7052, and classified it into the glycoside hydrolase family 39 (GH39). GAf...

The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)3 (Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains c...

β‐l‐arabinofuranosidases are thought to make use of a unique zinc‐associated cysteine nucleophile to catalyze glycoside hydrolysis. Using a bespoke synthetic covalent inhibitor, this proposed mechanism is put to the test. Mass spectrometry, X‐ray crystallography, and mechanistic simulations support the formation of a thioglycosyl enzyme intermediat...

C-Mannosyl tryptophan (CMW) is a unique glycosylated amino acid, and a candidate novel biomarker of renal function. In type 2 diabetes (T2D), a combination of metabolites including CMW has recently been the focus of novel biomarkers for the evaluation of renal function and prediction of its decline. However, previous quantification methods for seru...

The lectin chaperones calnexin (CNX) and calreticulin (CRT) localized in the endoplasmic reticulum play important roles in glycoprotein quality control. Although the interaction between these lectin chaperones and ERp57 is well known, it has been recently reported that endoplasmic reticulum protein 29 (ERp29), a member of PDI family, interacts with...

Activation of lysolipid-sensitive G protein-coupled receptors (GPCR) depends not only on lysolipid class but also on the length and degree of saturation of their respective hydrophobic tails. Positive regulation of these signaling networks caused by the lipid chain length specificity of upstream phospholipases is firmly established. Nonagonistic ly...

Rhodopsin-like G protein-coupled receptor (GPCR) GPR55 is attracting attention as a pharmaceutical target, because of its relationship with various physiological and pathological events. Although GPR55 was initially deorphanized as a cannabinoid receptor, lysophosphatidylinositol (LPI) is now widely perceived to be an endogenous ligand of GPR55. Re...

C-Mannosyl tryptophan (C-Man-Trp) is a unique glycosylated amino acid present in various eukaryotes. The C-Man-Trp structure can be found as a monomeric form or a part of post-translational modifications within polypeptide chains in living organisms. However, the mechanism of how monomeric C-Man-Trp is produced has not been fully investigated. In t...

The introduction of Asn‐linked glycans to nascent polypeptides occurs in the lumen of the endoplasmic reticulum of eukaryotic cells. After the removal of specific sugar residues, glycoproteins acquire signals in the glycoprotein quality control (GPQC) system and enter the folding cycle composed of lectin‐chaperones calnexin (CNX) and calreticulin (...

Background In the endoplasmic reticulum (ER), folding of glycoproteins is assisted by a combined action of enzymes and chaperones that leads them to biologically functional structures. In this system, UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) plays an essential role as the “folding sensor” by virtue of its ability to discriminate foldi...

Glycosphingolipid (GSL) is a group of molecules composed of hydrophilic glycan part and hydophobic ceramide creating diverse family. GSLs are de novo synthesised from ceramide at endoplasmic reticulum and Golgi apparatus, and transported to outer surface of plasma membrane. It has been known that the glycan structures of GSLs changes reflecting dis...

Calnexin (CNX) and calreticulin (CRT) are ER-resident lectin-like molecular chaperones involved in the quality control of secretory or membrane glycoproteins. They can exert molecular chaperone functions via specific binding to the early processing intermediates of Glc1Man9GlcNAc2 oligosaccharides of N-glycoproteins. CNX and CRT have similar N-term...

De‐glucosylation and re‐glucosylation of glycoproteins by glucosidase II and uridine diphosphate‐glucose: glycoprotein glucosyltransferase 1 (UGGT1), respectively, are important steps in glycoprotein quality control. Misfolded glycoprotein accumulation is associated with endoplasmic reticulum stress and can lead to protein misfolding diseases such...

Polymers of glucose, the most abundant and one of the biologically important natural product, named glucans, are widely present in fungal, bacterial, mammalian and plant with various anomeric configurations and glycosidic linkages. Because of their structural diversity, unified strategy for the assembly of pure glucans is yet to be developed. Herei...

Methyl 5-thio-α-d-mannopyranoside (1) and six inositols were evaluated for their ability to bind to pradimicins (PRMs) via molecular modeling and three binding assays. In all the experiments, the binding affinity of 1 was slightly lower than that of methyl α-d-mannopyranoside (Man-OMe) and inositols hardly bound to PRMs. These results indicate that...

Oxidative folding of proinsulin in the endoplasmic reticulum (ER) is critical for the proper sorting and secretion of insulin from pancreatic β-cells. Here, by using non-cell-based insulin aggregation assays and mouse insulinoma-derived MIN6 cells, we searched for a candidate molecular chaperone for (pro)insulin when its oxidative folding is compro...

A highly unique type of protein glycosylation was identified, in which a mannose residue is linked to tryptophan as C-glycoside. C-Mannosyl tryptophan (CMW) has been discovered in various proteins, such as RNase 2, complements, properdin, thrombospondin, F-spondin, erythropoietin receptor, mucins, ADAMTSs, and myelin-associated glycoprotein. Howeve...

Ovarian cancer survival is poor, in part, because there are no specific biomarkers for early diagnosis. C-Mannosyl tryptophan (CMW) is a structurally unique glycosylated amino acid recently identified as a novel biomarker of renal dysfunction. The present study investigated whether blood CMW is altered in patients with ovarian cancer and whether di...

Oligosaccharide, Glycoconjugates, Glycosylation, Stereoselectivity

Glycan engineering of antibody has received considerable attention. Although various endo-β-N-acetylglucosaminidase mutants have been developed for glycan remodeling, side-reaction has been reported between glycan oxazoline and amino groups. In this study, we performed a detailed characterization for antibody products obtained through enzymatic and...

Naturally occurring pradimicins (PRMs) show specific recognition of d-mannose (d-Man) in aqueous media, which has never been achieved by artificial small molecules. Although the Ca2+-mediated dimerization of PRMs is essential for their d-Man binding, the dimeric structure has yet to be elucidated, leaving the question open as to how PRMs recognize...

C-Mannosyl tryptophan (C-Man-Trp) is a unique molecule in that an α-mannose is connected to the indole C2 carbon atom of a Trp residue via C-glycosidic linkage. Although serum C-Man-Trp may be a novel biomarker of renal function in humans, the biological significance of C-Man-Trp has yet to be fully investigated. In this study, a novel assay system...

The glycoprotein quality control (GQC) system in the endoplasmic reticulum (ER) effectively uses chaperone-type enzymes and lectins such as UDP-glucose:glycoprotein glucosyltransferase (UGGT), calnexin (CNX), calreticulin (CRT), protein di-sulfide bond isomerases (ERp57 or PDIs), and glucosidases to generate native-folded glycoproteins from nascent...

G protein-coupled receptor 55 (GPR55) is highly expressed in brain and peripheral nervous system. Originally de-orphanized as a cannabinoid receptor, recently GPR55 has been described as a lysophospholipid responsive receptor, specifically towards lysophosphatidylinositol and lysophosphatidyl-β-D-glucoside (LysoPtdGlc). To characterize lysolipid-GP...

N-acetylglucosaminyltransferase-V (GnT-V) alters the structure of specific N-glycans by modifying α1-6-linked mannose with a β1-6-linked N-acetylglucosamine branch. β1-6 branch formation on cell surface receptors accelerates cancer metastasis, making GnT-V a promising target for drug development. However, the molecular basis of GnT-V's catalytic me...

We report a novel strategy for obtaining both anomers from a single mannosyl donor equipped with a C2-o-TsNHbenzyl ether (2-O-TAB) by switching reaction conditions. In particular, the formation of various β-mannosides was achieved with high selectivity by using a mannosyl phosphite in the presence of ZnI2.

A glucosyl donor equipped with C2-o-TsNHbenzyl ether was shown to provide both α- and β-glycosides stereoselectivity, by changing the reaction conditions. Namely, β-glycosides were selectively obtained when the trichloroacetimidate was activated by Tf2NH. On the other hand, activation by TfOH in Et2O provided α-glycosides as major products. This “s...

Lysophosphatidyl glucoside (LPGlc) is a structurally unique glycolipid that acts as a guidance cue for extending axons during central nervous system development by activating the class A G protein coupled receptor (GPR) 55 of spinal cord sensory axons. GPR55 not only plays an important role during development, but is also implicated in many disease...

The endo-β-N-acetylglucosaminidase mutant endo-CC N180H transfers glycan from sialylglycopeptide (SGP) to various acceptors. The scope and limitations of low-molecular-weight acceptors were investigated. Several homogeneous glycan-containing compounds, especially those with potentially useful labels or functional moieties, and possible reagents in...

Acceptor range of endo-β-N-acetylglucosaminidase Mutant endo-CC N180H: from Monosaccharide to Antibody: Supporting information

C-Mannosylation, a protein-modification found in various eukaryotes, involves the attachment of a single mannose molecule to selected tryptophan residues of proteins. Since C-mannosyl tryptophan (CMW) was detected in human urine, it is generally thought that CMW is not catabolized inside our body and instead is excreted via the urine. This paper re...

Glycosides can be cleaved in either an exo- or endocyclic fashion. Specifically, the former pathway is used during conventional glycosylation; whereas the latter pathway has not been the subject of attention. Endocyclic cleavage reaction enable the anomerization of β-glycosides to α-glycosides. Herein, we compared the efficacies of endo- and exocyc...

Glycan chains of various glycoconjugates are known to play pivotal roles in biological systems. Given their structural complexity diversity, chemical synthesis is expected to be powerful in understanding functions of glycan chains at molecular level. However, synthesis of glycan chains is more problematic than other biomolecules such as oligopeptid...

A series of compounds associated to naturally occurring and biologically relevant glycans consisting of α-mannosides were prepared and analyzed using collision-induced dissociation (CID), energy-resolved mass spectrometry (ERMS), and 1H nuclear magnetic resonance spectroscopy. The CID experiments of sodiated species of disaccharides and ERMS experi...

Orexin neurons regulate critical brain activities for controlling sleep, eating, emotions, and metabolism, and impaired orexin neuron function results in several neurological disorders. Therefore, restoring normal orexin function and understanding the mechanisms of loss or impairment of orexin neurons represent important goals. As a step toward tha...

UDP-glucose:glycoprotein glucosyltransferase (UGGT) distinguishes glycoproteins in non-native conformations from those in native conformations, and glucosylates only non-native glycoproteins. To analyze how UGGT recognizes non-native glycoproteins, we chemically synthesized site-specifically 15N-labeled interleukin 8 (IL-8) C-terminal (34–72) glyco...

The inside cover picture shows a transglycosylation reaction catalyzed by Golgi endo-α-mannosidase (G-EM). We have demonstrated that G-EM hydrolysis activity can be employed to mediate transglycosylation by using α-fluorinated glucosyl mannoside as a donor. Systematic screening of various point mutants revealed that the E407D mutant had excellent t...

This volume focuses on solution and solid-state NMR of carbohydrates, glycoproteins, glyco-technologies, biomass and related topics. It is estimated that at least 80% of all proteins are glycoproteins. Because of the complexity, heterogeneity and flexibility of the sugar chains, the structural biology approaches for glycoconjugates have been genera...

The inside cover picture shows inhibitor-based manipulation of glycoprotein sorting. Several α-1,2-mannosidases in the endoplasmic reticulum (ER) play important roles in producing signal glycoforms that control glyocprotein fate. With 1-deoxymannojirimycin (dMJ), the glycan was selectively trimmed to the secretion signal in the ER fraction. On the...

The enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) mediates quality control of glycoproteins in the endoplasmic reticulum by attaching glucose to N-linked glycan of misfolded proteins. As a sensor, UGGT ensures that misfolded proteins are recognized by the lectin chaperones and do not leave the secretory pathway. The structure of UGGT a...

In order for facilitating the synthesis of oligosaccharides, transglycosylation reactions mediated by glycoside hydrolases have been studied in various contexts. In this study, we examined transglycosylating activity of Golgi endo-α-mannosidase. To this end, we prepared various glycosyl donors and acceptors, and recombinant human Golgi endo-α-manno...

Endoplasmic reticulum (ER) resident lectin chaperone calnexin (CNX) and calreticulin (CRT) assist folding of nascent glycoproteins. Their association with ERp57, a member of PDI family proteins (PDIs) which promote disulfide bond formation of unfolded proteins, has been well documented. Recent studies have provided evidence that other PDIs may also...

Within the endoplasmic reticulum immature glycoproteins are sorted to secretion and degradation pathways through the sequential trimming of mannose residues from Man9GlcNAc2 to Man5GlcNAc2 by the combined actions of assorted ?-1,2-mannosidases. It has been speculated that specific glycoforms encode signals for secretion and degradation. However, it...

The intramolecular aglycon delivery (IAD) methodology through various mixed acetal linkages has been extensively studied, due to its potential to provide a solution for stereoselective 1,2-cis glycoside formations. Stereoselectivity of glycosylation through IAD may well be independent of the structure of the acceptor allowing the exclusive formatio...

Glycoprotein N-linked oligosaccharides in the endoplasmic reticulum function as tags to regulate glycoprotein folding, sorting, secretion and degradation. Since the N-glycan structure of a glycoprotein should reflect the folding state, N-glycan processing may be affected by the aglycone state. In this study, we examined the influence of aglycone st...

Recently, a number of chemoenzymatic strategies have been explored for achieving preparation of homogeneous glycopeptides and glycoproteins, especially by using endoglycanases and glycosyl oxazolines. However, concomitant occurrence of non-enzymatic reactions has been reported, but no further characterization of the byproducts was conducted. In thi...