Synthesis of Sucrose-Mimicking Disaccharide by Intramolecular Aglycone Delivery.

Rare sugars are known for their ability to suppress postprandial blood glucose levels. Therefore, oligosaccharides and disaccharides derived from rare sugars could potentially serve as functional sweeteners. A disaccharide [α-d-allopyranosyl-(1→2)-ß-d-psicofuranoside] mimicking sucrose was synthesized from rare monosaccharides D-allose and D-psicose. Glycosylation using the intermolecular aglycon delivery (IAD) method was employed to selectively form 1,2-cis α-glycosidic linkages of the allopyranose residues. Moreover, ß-selective psicofuranosylation was performed using a psicofuranosyl acceptor with 1,3,4,6-tetra-O-benzoyl groups. This is the first report on the synthesis of non-reducing disaccharides comprising only rare d-sugars by IAD using protected ketose as a unique acceptor; additionally, this approach is expected to be applicable to the synthesis of functional sweeteners.

Molecular basis of N-glycan recognition by pradimicin a and its potential as a SARS-CoV-2 entry inhibitor.

Virus entry inhibitors are emerging as an attractive class of therapeutics for the suppression of viral transmission. Naturally occurring pradimicin A (PRM-A) has received particular attention as the first-in-class entry inhibitor that targets N-glycans present on viral surface. Despite the uniqueness of its glycan-targeted antiviral activity, there is still limited knowledge regarding how PRM-A binds to viral N-glycans. Therefore, in this study, we performed binding analysis of PRM-A with synthetic oligosaccharides that reflect the structural motifs characteristic of viral N-glycans. Binding assays and molecular modeling collectively suggest that PRM-A preferentially binds to branched oligomannose motifs of N-glycans via simultaneous recognition of two mannose residues at the non-reducing ends. We also demonstrated, for the first time, that PRM-A can effectively inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vitro. Significantly, the anti-SARS-CoV-2 effect of PRM-A is attenuated in the presence of the synthetic branched oligomannose, suggesting that the inhibition of SARS-CoV-2 infection is due to the interaction of PRM-A with the branched oligomannose-containing N-glycans. These data provide essential information needed to understand the antiviral mechanism of PRM-A and suggest that PRM-A could serve as a candidate SARS-CoV-2 entry inhibitor targeting N-glycans.

Bifidobacterial GH146 ß-L-arabinofuranosidase for the removal of ß1,3-L-arabinofuranosides on plant glycans.

L-Arabinofuranosides with ß-linkages are present in several plant molecules, such as arabinogalactan proteins (AGPs), extensin, arabinan, and rhamnogalacturonan-II. We previously characterized a ß-L-arabinofuranosidase from Bifidobacterium longum subsp. longum JCM 1217, Bll1HypBA1, which was found to belong to the glycoside hydrolase (GH) family 127. This strain encodes two GH127 genes and two GH146 genes. In the present study, we characterized a GH146 ß-L-arabinofuranosidase, Bll3HypBA1 (BLLJ_1848), which was found to constitute a gene cluster with AGP-degrading enzymes. This recombinant enzyme degraded AGPs and arabinan, which contain Araf-ß1,3-Araf structures. In addition, the recombinant enzyme hydrolyzed oligosaccharides containing Araf-ß1,3-Araf structures but not those containing Araf-ß1,2-Araf and Araf-ß1,5-Araf structures. The crystal structures of Bll3HypBA1 were determined at resolutions up to 1.7 Å. The monomeric structure of Bll3HypBA1 comprised a catalytic (α/α)6 barrel and two ß-sandwich domains. A hairpin structure with two ß-strands was observed in Bll3HypBA1, to extend from a ß-sandwich domain and partially cover the active site. The active site contains a Zn2+ ion coordinated by Cys3-Glu and exhibits structural conservation of the GH127 cysteine glycosidase Bll1HypBA1. This is the first study to report on a ß1,3-specific ß-L-arabinofuranosidase. KEY POINTS: • ß1,3-l-Arabinofuranose residues are present in arabinogalactan proteins and arabinans as a terminal sugar. • ß-l-Arabinofuranosidases are widely present in intestinal bacteria. • Bll3HypBA1 is the first enzyme characterized as a ß1,3-linkage-specific ß-l-arabinofuranosidase.

Identification and characterization of endo-α-, exo-α-, and exo-ß-D-arabinofuranosidases degrading lipoarabinomannan and arabinogalactan of mycobacteria.

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 mammalian immune responses. In this study, we identified four enzymes (two GH183 endo-D-arabinanases, GH172 exo-α-D-arabinofuranosidase, and GH116 exo-ß-D-arabinofuranosidase) from Microbacterium arabinogalactanolyticum. These enzymes completely degraded the complex D-arabinan core structure of lipoarabinomannan and arabinogalactan in a concerted manner. Furthermore, through biochemical characterization using synthetic substrates and X-ray crystallography, we elucidated the mechanisms of substrate recognition and anomer-retaining hydrolysis for the α- and ß-D-arabinofuranosidic bonds in both endo- and exo-mode reactions. The discovery of these D-arabinan-degrading enzymes, along with the understanding of their structural basis for substrate specificity, provides valuable resources for investigating the intricate glycan architecture of mycobacterial cell wall polysaccharides and their contribution to pathogenicity.

Recent Progress in 1,2-cis glycosylation for Glucan Synthesis.

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 ß-mannoside in glycoproteins, glycolipids, proteoglycans, microbial polysaccharides, and bioactive natural products. In the structure of polysaccharides such as α-glucan, 1,2-cis α-glucosides were found to be the major linkage between the glucopyranosides. Various regioisomeric linkages, 1→3, 1→4, and 1→6 for the backbone structure, and 1→2/3/4/6 for branching in the polysaccharide as well as in the oligosaccharides were identified. To achieve highly stereoselective 1,2-cis glycosylation, including α-glucosylation, a number of strategies using inter- and intra-molecular methodologies have been explored. Recently, Zn salt-mediated cis glycosylation has been developed and applied to the synthesis of various 1,2-cis linkages, such as α-glucoside and ß-mannoside, via the 1,2-cis glycosylation pathway and ß-galactoside 1,4/6-cis induction. Furthermore, the synthesis of various structures of α-glucans has been achieved using the recent progressive stereoselective 1,2-cis glycosylation reactions. In this review, recent advances in stereoselective 1,2-cis glycosylation, particularly focused on α-glucosylation, and their applications in the construction of linear and branched α-glucans are summarized.

Squaryl group-modified UDP analogs as inhibitors of the endoplasmic reticulum-resident folding sensor enzyme UGGT.

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 inhibitory activity are scarce. Here, we report squaryl group-modified UDP analogs as a promising UGGT inhibitor. Among these, the compound possessing a 2'-amino group of the uridine moiety and hydroxyethyl-substituted squaramide exhibited the highest potency, suggesting its relevance as a molecule for further optimization.

Bifidobacterial GH146 ß-l-Arabinofuranosidase (Bll4HypBA1) as the Last Enzyme for the Complete Removal of Oligoarabinofuranosides from Hydroxyproline-Rich Glycoproteins.

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)-ß-l-Araf-(1→2)-ß-l-Araf-(1→2)-ß-l-Araf, l-Araf4 ), whose linkages are targets of the bifidobacterial and Xanthomonas arabinooligosaccharide-degrading enzymes. The l-Araf4 motif was cleaved by GH43 α-l-arabinofuranosidase (Arafase) and converted to an l-Araf3 -linked structure. The latter is then cleaved by GH121 ß-l-arabinobiosidase (HypBA2), producing ß-l-Araf-(1→2)-l-Ara (ß-l-arabinobiose) and mono-ß-l-Araf linked to the HRGP backbone. In bifidobacteria, the ß-l-arabinobiose is then hydrolyzed by GH127 ß-l-Arafase (Bll1HypBA1), a mechanistically unique cysteine glycosidase. We recently identified the distantly related homologue from Xanthomonas euvesicatoria as GH146 ß-l-Arafase along with paralogues from Bifidobacterium longum, one of which, Bll4HypBA1 (BLLJ_0089), can degrade l-Araf1 -Hyp in a similar way to that of GH146. As the chemical synthesis of the extensin hydrophilic motif 1 a, which possesses three distinct linkages that connect four oligoAraf residues [Hyp(l-Arafn ) (n=4, 3, 1)], was achieved previously, we precisely monitored the step-wise enzymatic cleavage of 1 a in addition to that of potato lectin. The results unequivocally revealed that this enzyme specifically degrades the Hyp(l-Araf1 ) motif.

Analysis of Selenoprotein F Binding to UDP-Glucose:Glycoprotein Glucosyltransferase (UGGT) by a Photoreactive Crosslinker.

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 human UGGT1 (HUGT1) revealed that SelenoF binds to the TRXL2 domain of HUGT1. Additionally, a newly developed photoaffinity crosslinker was selectively introduced into cysteine residues of recombinant SelenoF to determine the spatial orientation of SelenoF to HUGT1. The crosslinking experiments showed that SelenoF formed a covalent bond with amino acids in the TRXL3 region and the interdomain between ßS2 and GT24 of HUGT1 via the synthetic crosslinker. SelenoF might play a role in assessing and refining the disulfide bonds of misfolded glycoproteins in the hydrophobic cavity of HUGT1 as it binds to the highly flexible region of HUGT1 to reach its long hydrophobic cavity. Clarification of the SelenoF-binding domain of UGGT and its relative position will help predict and reveal the function of SelenoF from a structural perspective.

Hidetoshi Yamada: His journey in the carbohydrate world.

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 prominent achievements in carbohydrate chemistry.

Mechanism-based inhibition of GH127/146 cysteine glycosidases by stereospecifically functionalized l-arabinofuranosides.

To understand the precise mechanism of the glycoside hydrolase (GH) family 127, a cysteine ß-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 derivative was used as the monoglycosylamine equivalent to afford the l-Araf-chloroacetamides (α/ß-1-Cl) as well as bromoacetamides (α/ß-1-Br) in highly stereoselective manner through Staudinger reaction followed by amide formation with/without anomerization. Against HypBA1, the probes 1, especially in the case of α/ß-1-Br inhibited the hydrolysis. Conformational implications of these observations are discussed in this manuscript. Additional examinations using l-Araf-azides (α/ß-5) resulted in further mechanistic observations of the GH127/146 cysteine glycosidases, including the hydrolysis of ß-5 as the substrate and oxidative inhibition by α-5 using the GH127 homologue.

Phenotypic analysis of α1,2-mannosidase-like protein deletion mutants in Saccharomyces cerevisiae.

α1,2­mannosidase-like proteins mediate quality control of glycoproteins in the endoplasmic reticulum. This study explored α1,2­mannosidase-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 growth rate was recovered in double-deletion strains where one of the deletions was in MNS1 . We also measured the mannosidase activity of microsomal fractions of deficient strains using artificial glycan. Increased mannose trimming activities were demonstrated by the microsomes of MNL2 -deletion strains compared to levels of activity exhibited by the microsomes of the control strain.

Recent advances in stereoselective 1,2-cis-O-glycosylations.

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, ß-mannopyranoside, ß-arabinofuranoside, and other rather rare glycosides are found in natural glycans, including glycoconjugate (glycoproteins, glycolipids, proteoglycans, and microbial polysaccharides) and glycoside natural products. The way to 1,2-trans isomers is well sophisticated by using the effect of neighboring group participation from the most effective and kinetically favored C-2 substituent such as an acyl group, although high stereoselective synthesis of 1,2-cis glycosides without formation of 1,2-trans isomers is far less straightforward. Although the key factors that control the stereoselectivity of glycosylation are largely understood since chemical glycosylation was considered to be one of the useful methods to obtain glycosidic linkages as the alternative way of isolation from natural sources, strictly controlled formation of these 1,2-cis glycosides is generally difficult. This minireview introduces some of the recent advances in the development of 1,2-cis selective glycosylations, including the quite recent developments in glycosyl donor modification, reaction conditions, and methods for activation of intermolecular glycosylation, including the bimodal glycosylation strategy for 1,2-cis and 1,2-trans glycosides, as well as intramolecular glycosylations, including recent applications of NAP-ether-mediated intramolecular aglycon delivery.

Synthesis of naturally occurring ß-l-arabinofuranosyl-l-arabinofuranoside structures towards the substrate specificity evaluation of ß-l-arabinofuranosidase.

Methyl ß-l-arabinofuranosyl-(1 â†’ 2)-, -(1 â†’ 3)-, and -(1 â†’ 5)-α-l-arabinofuranosides have been stereoselectively synthesized through 2-naphthylmethyl ether-mediated intramolecular aglycon delivery (NAP-IAD), whose ß-linkages were confirmed by NMR analysis on the 3JH1-H2 coupling constant and 13C chemical shift of C1. The NAP-IAD approach was simply extended for the synthesis of trisaccharide motifs possessing ß-l-arabinofuranosyl-(1 â†’ 5)-l-arabinofuranosyl non-reducing terminal structure with the branched ß-l-arabinofuranosyl-(1 â†’ 5)-[α-l-arabinofuranosyl-(1 â†’ 3)]-α-l-arabinofuranosyl and the liner ß-l-arabinofuranosyl-(1 â†’ 5)-ß-l-arabinofuranosyl-(1 â†’ 5)-ß-l-arabinofuranosyl structures in olive arabinan and dinoflagellate polyethers, respectively. The results on the substrate specificity of a bifidobacterial ß-l-arabinofuranosidase HypBA1 using the regioisomers indicated that HypBA1 could hydrolyze all three linkages however behaved clearly less active to ß-(1 â†’ 5)-linked disaccharide than other two regioisomers including the proposed natural degradation product, ß-(1 â†’ 2)-linked one from plant extracellular matrix such as extensin. On the other hand, Xanthomonas XeHypBA1 was found to hydrolyze all three disaccharides as the substrate with higher specificity to ß-(1 â†’ 2)-linkage than bifidobacterial HypBA1.

TMED3 Complex Mediates ER Stress-Associated Secretion of CFTR, Pendrin, and SARS-CoV-2 Spike.

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 transmembrane proteins. The gene silencing results reveal that TMED2, TMED3, TMED9 and TMED10 are involved in the UPS of transmembrane proteins, such as CFTR, pendrin and SARS-CoV-2 Spike. Subsequent mechanistic analyses indicate that TMED3 recognizes the ER core-glycosylated protein cargos and that the heteromeric TMED2/3/9/10 complex mediates their UPS. Co-expression of all four TMEDs improves, while each single expression reduces, the UPS and ion transport function of trafficking-deficient ΔF508-CFTR and p.H723R-pendrin, which cause cystic fibrosis and Pendred syndrome, respectively. In contrast, TMED2/3/9/10 silencing reduces SARS-CoV-2 viral release. These results provide evidence for a common role of TMED3 and related TMEDs in the ER stress-associated, Golgi-independent secretion of transmembrane proteins.

Recent Chemical and Chemoenzymatic Strategies to Complex-Type N-Glycans.

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 analytical or medicinal purposes, efficient and universal methods to provide homogeneous complex-type N-glycans have been an urgent need. Despite the extremely complicated structures, tremendous progress in the synthesis of N-glycans has been achieved. On one hand, chemical strategies are shown to be effective to prepare core oligosaccharides of N-glycans by focusing on stereoselective glycosylations such as ß-mannosylation and α-sialylation, as well as the methodology of the N-glycan assembly. On the other hand, chemoenzymatic strategies have also become increasingly powerful in recent years. This review attempts to highlight the very recent advancements in chemical and chemoenzymatic strategies for eukaryotic complex-type N-glycans.

In vitro mannosidase activity of EDEM3 against asparagine-linked oligomannose-type glycans.

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 responsible for mannose trimming. However, the exact role of EDEMs as α-mannosidases in ERAD remains unclear. Here, we performed the biochemical characterization of EDEM3 using synthetic oligomannose-type glycan substrates. In vitro assays revealed that EDEM3 can convert an asparagine-linked M9 glycan to M8 and M7 glycans in contrast to glycine-linked M9 glycan, and the activity is enhanced in the presence of ERp46, a known partner protein of EDEM3. Our study provides novel insights into the enzymatic properties of EDEM3 and the use of artificial glycan substrates as tools to study ERAD mechanisms.

Mechanism of Cooperative Degradation of Gum Arabic Arabinogalactan Protein by Bifidobacterium longum Surface Enzymes.

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. longum. The enzyme released α-d-Galp-(1→3)-l-Ara and ß-l-Arap-(1→3)-l-Ara from gum arabic AGP and facilitated the action of other enzymes for degrading the AGP backbone and modified sugar. In this study, we identified an α-l-arabinofuranosidase (BlArafE; encoded by BLLJ_1850), a multidomain enzyme with both GH43_22 and GH43_34 catalytic domains, as a critical enzyme for the degradation of modified α-l-arabinofuranosides in gum arabic AGP. Site-directed mutagenesis approaches revealed that the α1,3/α1,4-Araf double-substituted gum arabic AGP side chain was initially degraded by the GH43_22 domain and subsequently cleaved by the GH43_34 domain to release α1,3-Araf and α1,4-Araf residues, respectively. Furthermore, we revealed that a tetrasaccharide, α-l-Rhap-(1→4)-ß-d-GlcpA-(1→6)-ß-d-Galp-(1→6)-d-Gal, was a limited degradative oligosaccharide in the gum arabic AGP fermentation of B. longum subsp. longum JCM7052. The oligosaccharide was produced from gum arabic AGP by the cooperative action of the three cell surface-anchoring enzymes, GAfase, exo-ß1,3-galactanase (Bl1,3Gal), and BlArafE, on B. longum subsp. longum JCM7052. Furthermore, the tetrasaccharide was utilized by the commensal bacteria. IMPORTANCE Terminal galactose residues of the side chain of gum arabic arabinogalactan protein (AGP) are mainly substituted by α1,3/α1,4-linked Araf and ß1,6-linked α-l-Rhap-(1→4)-ß-d-GlcpA residues. This study found a multidomain BlArafE with GH43_22 and GH43_34 catalytic domains showing cooperative action for degrading α1,3/α1,4-linked Araf of the side chain of gum arabic AGP. In particular, the GH43_34 domain of BlArafE was a novel α-l-arabinofuranosidase for cleaving the α1,4-Araf linkage of terminal galactose. α-l-Rhap-(1→4)-ß-d-GlcpA-(1→6)-ß-d-Galp-(1→6)-d-Gal tetrasaccharide was released from gum arabic AGP by the cooperative action of GAfase, GH43_24 exo-ß-1,3-galactanase (Bl1,3Gal), and BlArafE and remained after B. longum subsp. longum JCM7052 culture. Furthermore, in vitro assimilation test of the remaining oligosaccharide using Bacteroides species revealed that cross-feeding may occur from bifidobacteria to other taxonomic groups in the gut.

Mannose-binding analysis and biological application of pradimicins.

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-insoluble aggregates. Recently, we have established a derivatization strategy to suppress PRM aggregation, providing an opportunity for practical application of PRMs in glycobiological research. This article first outlines the challenges in studying Man-binding mechanisms and structural modifications of PRMs, and then describes our approach to address them. We also present our recent attempts toward the development of PRM-based research tools.

C-Mannosylated tryptophan-containing WSPW peptide binds to actinin-4 and alters E-cadherin subcellular localization in lung epithelial-like A549 cells.

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 remain to be elucidated. By using chemically synthesized WSPW peptides and C-mannosylated WSPW peptides (C-Man-WSPW), we herein investigated whether C-mannosylation of WSPW peptides confer additional biological functions to WSPW peptides. C-Man-WSPW peptide, but not non-mannosylated WSPW, reduced E-cadherin levels in A549 cells. Via peptide mass fingerprinting analysis, we identified actinin-4 as a C-Man-WSPW-binding protein in A549 cells. Actinin-4 partly co-localized with E-cadherin or ß-catenin, despite no direct interaction between actinin-4 and E-cadherin. C-Man-WSPW reduced co-localization of E-cadherin and actinin-4; non-mannosylated WSPW had no effect on localization. In actinin-4-knockdown cells, E-cadherin was upregulated and demonstrated a punctate staining pattern in the cytoplasm, which suggests that actinin-4 regulated cell-surface E-cadherin localization. Thus, C-mannosylation of WSPW peptides is required for interaction with actinin-4 that subsequently alters expression and subcellular localization of E-cadherin and morphology of epithelial-like cells. Our results therefore suggest a regulatory role of C-mannosylation of the W-x-x-W motif in interactions between the motif and its binding partner and will thereby enhance understanding of protein C-mannosylation.