Glycoproteins in non-native conformations are often toxic to cells and may cause diseases, thus the quality control (QC) system eliminates these unwanted species. Lectin chaperone calreticulin and glucosidase II, both of which recognize the Glc₁Man₉ oligosaccharide on glycoproteins, are important components of the glycoprotein QC system. Reported herein is the preparation of Glc₁Man₉-glycoproteins in both native and non-native conformations by using the following sequence: misfolding of chemically synthesized Man₉-glycoprotein, enzymatic glucosylation, and another misfolding step. By using synthetic glycoprotein probes, calreticulin was found to bind preferentially to a hydrophobic non-native glycoprotein whereas glucosidase II activity was not affected by glycoprotein conformation. The results demonstrate the ability of chemical synthesis to deliver homogeneous glycoproteins in several non-native conformations for probing the glycoprotein QC system.

Glycoproteins in non-native conformations are often toxic to cells and may cause diseases, thus the quality control (QC) system eliminates these unwanted species. Lectin chaperone calreticulin and glucosidase II, both of which recognize the Glc₁Man₉ oligosaccharide on glycoproteins, are important components of the glycoprotein QC system. Reported herein is the preparation of Glc₁Man₉-glycoproteins in both native and non-native conformations by using the following sequence: misfolding of chemically synthesized Man₉-glycoprotein, enzymatic glucosylation, and another misfolding step. By using synthetic glycoprotein probes, calreticulin was found to bind preferentially to a hydrophobic non-native glycoprotein whereas glucosidase II activity was not affected by glycoprotein conformation. The results demonstrate the ability of chemical synthesis to deliver homogeneous glycoproteins in several non-native conformations for probing the glycoprotein QC system.

Asparagine-linked (N-linked) sugar chains are widely found in the rough endoplasmic reticulum (ER), which has attracted renewed attention because of its participation in the glycoprotein quality control process. In the ER, newly formed glycoproteins are properly folded to higher-order structures by the action of a variety of lectin chaperones and processing enzymes and are transported into the Golgi, while terminally misfolded glycoproteins are carried into the cytosol for degradation. A group of proteins related to this system are known to recognize subtle differences in the high-mannose-type oligosaccharide structures of glycoproteins; however, their molecular foundations are still unclear. In order to gain a more precise understanding, our group has established a strategy for the systematic synthesis of high-mannose-type glycans. More recently, we have developed “top-down” chemoenzymatic approaches that allow expeditious access to theoretically all types of high-mannose glycans. This strategy comprehensively delivered 37 high-mannose-type glycans, including G1M9–M3 glycans, and opened up the possibility of the elucidation of structure–function relationships with a series of high-mannose-type glycans.

A comprehensive method for the construction of a high-mannose-type glycan library by systematic chemo-enzymatic trimming of a single Man9-based precursor was developed. It consists of the chemical synthesis of a non-natural tridecasaccharide precursor, the orthogonal demasking of the non-reducing ends, and trimming by glycosidases, which enabled a comprehensive synthesis of high-mannose-type glycans in their mono- or non-glucosylated forms. It employed glucose, isopropylidene, and N-acetylglucosamine groups for blocking the A-, B-, and C-arms, respectively. After systematic trimming of the precursor, thirty-seven high-mannose-type glycans were obtained. The power of the methodology was demonstrated by the enzymatic activity of human recombinant N-acetylglucosaminyltransferase-I toward M7–M3 glycans, clarifying the substrate specificity in the context of high-mannose-type glycans.

The oligosaccharyltransferase PglB from Campylobacter jejuni catalyses the N-glycosylation reaction with undecaprenyl-pyrophosphate-linked Glc₁GalNAc₅Bac₁ (Und-PP-Glc₁GalNAc₅Bac₁). Experiments using chemically synthesized donors coupled to fluorescently tagged peptides confirmed that biosynthetic intermediate Und-PP-Bac₁ and Und-PP-GalNAc₂Bac₁ are transferred efficiently to the Asn residue in the consensus sequence (D/E-X′-N-X-T/S, X′,X≠P). The products were analyzed in detail by tandem MS to confirm their chemical structures.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) plays a key role in recognizing folded and misfolded glycoproteins in the glycoprotein quality control system of the endoplasmic reticulum. UGGT detects misfolded glycoproteins and re-glucosylates them as a tag for misfolded glycoproteins. A flexible model to reproduce in vitro folding of a glycoprotein in the presence of UGGT in a mixture containing correctly folded, folding intermediates, and misfolded glycoproteins is described. The data demonstrates that UGGT can re-glucosylate all intermediates in the in vitro folding experiments, thus indicating that UGGT inspects not only final folded products, but also the glycoprotein folding intermediates.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) plays a key role in recognizing folded and misfolded glycoproteins in the glycoprotein quality control system of the endoplasmic reticulum. UGGT detects misfolded glycoproteins and re-glucosylates them as a tag for misfolded glycoproteins. A flexible model to reproduce in vitro folding of a glycoprotein in the presence of UGGT in a mixture containing correctly folded, folding intermediates, and misfolded glycoproteins is described. The data demonstrates that UGGT can re-glucosylate all intermediates in the in vitro folding experiments, thus indicating that UGGT inspects not only final folded products, but also the glycoprotein folding intermediates.

Extensin, the structural motif of plant extracellular matrix proteins, possesses a unique highly glycosylated, hydrophilic, and repeating Ser₁Hyp₄ pentapeptide unit, and has been proposed to include post-translational hydroxylation at proline residue and subsequent oligo-L-arabinosylations at all of the resultant hydroxyprolines as well as galactosylation at serine residue. Reported herein is the stereoselective synthesis of one of the highly glycosylated motifs, Ser(Galp₁)-Hyp(Araf₄)-Hyp(Araf₄)-Hyp(Araf₃)-Hyp(Araf₁). The synthesis has been completed by the application of 2-(naphthyl)methylether-mediated intramolecular aglycon delivery to the stereoselective construction of the Ser(Galp₁) and Hyp(Araf_n) fragments as the key step, as well as Fmoc solid-phase peptide synthesis for the backbone pentapeptide.

Pradimicins (PRMs) and benanomicins are the only family of non-peptidic natural products with lectin-like properties, that is, they recognize D-mannopyranoside (Man) in the presence of Ca²⁺ ions. Coupled with their unique Man binding ability, they exhibit antifungal and anti-HIV activities through binding to Man-containing glycans of pathogens. Notwithstanding the great potential of PRMs as the lectin mimics and therapeutic leads, their molecular basis of Man recognition has yet to be established. Their aggregate-forming propensity has impeded conventional interaction analysis in solution, and the analytical difficulty is exacerbated by the existence of two Man binding sites in PRMs. In this work, we investigated the geometry of the primary Man binding of PRM-A, an original member of PRMs, by the recently developed analytical strategy using the solid aggregate composed of the 1:1 complex of PRM-A and Man. Evaluation of intermolecular distances by solid-state NMR spectroscopy revealed that the C2–C4 region of Man is in close contact with the primary binding site of PRM-A, while the C1 and C6 positions of Man are relatively distant. The binding geometry was further validated by co-precipitation experiments using deoxy-Man derivatives, leading to the proposal that PRM-A binds not only to terminal Man residues at the non-reducing end of glycans, but also to internal 6-substituted Man residues. The present study provides new insights into the molecular basis of Man recognition and glycan specificity of PRM-A.

2,3-trans-carbamate- and -carbonate-carrying pyranosides were very easily anomerised from the β to the α direction in the presence of a Lewis acid compared to other pyranosides. This reaction is caused by endocyclic cleavage of the pyranosides. Evidence for endocyclic cleavage of conformationally restricted pyranosides in the chair form was obtained by intra- and intermolecular Friedel–Crafts reactions, chloride addition, and reduction of the generated cation. On the other hand, pyranosides with the distorted conformation were never cleaved in an endocyclic manner.