Masayuki Izumi

Expression of α2,8/2,9-Polysialyltransferase fromEscherichia coli K92 CHARACTERIZATION OF THE ENZYME AND ITS REACTION PRODUCTS

The capsular polysaccharide of Escherichia coli K92 contains alternating -8-NeuAcα2- and -9-NeuAcα2- linkages. The enzyme catalyzing this polymerizing reaction has been cloned from the genomic DNA of E. coli K92. The 1.2-kilobase polymerase chain reaction fragment was subcloned in pRSET vector and the protein was expressed in the BL21(DE3) strain ofE. coli with a hexameric histidine at its N-terminal end. The enzyme was isolated in the supernatant after lysis of the cells and fractionated by ultracentrifugation. Western blotting using anti-histidine antibody showed the presence of a band that migrated at about 47.5 kDa on both reducing and nonreducing SDS-polyacrylamide gel electrophoresis, indicating a monomeric enzyme. Among the carbohydrate acceptors tested, N-acetylneuraminic acid and the gangliosides GD3 and GQ1b were preferred substrates. The cell-free enzyme reaction products obtained were characterized by NMR and mass spectrometry, which indicated the presence of both α2,9- and α2,8-linked polysialyl structure. The K92 neuS gene was used to transform the K1 strain ofE. coli, the capsule of which contains only -8-NeuAcα2- linkages. Analysis of the polysaccharides isolated from these transformed cells is consistent with the presence of both -8-NeuAcα2- and -9-NeuAcα2- linkages. Our results suggest that theneuS gene product of E. coli K92 catalyzes the synthesis of polysialic acid with α2,9- and α2,8-linkages in vitro and in vivo.

Chemical Synthesis of Intentionally Misfolded Homogeneous Glycoprotein: A Unique Approach for the Study of Glycoprotein Quality Control

Biosynthesis of glycoproteins in the endoplasmic reticulum employs a quality control system, which discriminates and excludes misfolded malfunctional glycoproteins from a correctly folded one. As chemical tools to study the glycoprotein quality control system, we systematically synthesized misfolded homogeneous glycoproteins bearing a high-mannose type oligosaccharide via oxidative misfolding of a chemically synthesized homogeneous glycopeptide. The endoplasmic reticulum folding sensor enzyme, UDP-glucose:glycoprotein glucosyltransferase (UGGT), recognizes a specific folding intermediate, which exhibits a molten globule-like hydrophobic nature.

A tong-like fluorescence sensor for metal ions: perfect conformational switch of hinge sugar by pyrene stacking.

Carbohydrates are among the potential materials for molecular devices, since they are abundant natural resources. However, their rigidity has restricted their use for movable devices. Hinge sugars, 2,4-diamino-2,4-dideoxy-xylopyranosides, shed light on the use of carbohydrates as movable components, as demonstrated by the motion by which all four equatorial substituents can change to an axial orientation in synchronization with a chelation-driven 4C1-1C4 ring flip. In this study, we synthesized a tong-like metal ion sensor, 1,3-di-O-pyrenylmethylated hinge sugar (1), and its model compound, methyl 2,4-di-O-pyrenecarbonyl-xylopyranoside (2), to extend the abilities of hinge sugars as molecular components. From observations of the solvent-dependent conformational and fluorescent behavior of 2, we found that the pyrene stacking assists the 1C4 formation of xylopyranoside by 1.7 kcal mol(-1). We also found that compound 1 produced excimer fluorescence by chelation to Pt2+, Zn2+, Cd2+, Mg2+ or Mn2+, and unexpectedly by addition of acids. 1H NMR measurements ascribed this behavior to the 4C1-1C4 ring flip of hinge sugar in response to chelation or protonation at N2, and revealed rapid and perfect 1C4 formation in the case of Zn2+. These findings will extend the scope of hinge sugars as movable components.

Both Isoforms of Human UDP-glucose:glycoprotein Glucosyltransferase are Enzymatically Active

Being recognized as an important constituent of the glycoprotein folding cycle, uridine diphosphate-glucose:glycoprotein glucosyltransferase (UGGT) has been a subject of intense study. Up to now, it is two isoforms, UGGT1 and 2 have been identified, which share ∼ 50% amino acid identity. UGGT1 is a well-documented enzyme which functions as a folding sensor in the endoplasmic reticulum, by the virtue of its ability to transfer a glucose residue to non-glucosylated high-mannose-type glycans of immature glycoproteins exhibiting non-native conformation. On the other hand, direct evidence to support the glucosyltransferase activity of UGGT2 has been lacking, leaving it unclear as to whether it has any function in the glycoprotein folding process. This study aimed to reveal the property of human UGGT2 by using synthetic substrates such as fluorescently labeled glycans and N-glycosylated proteins. The analysis, for the first time, revealed the glucosyltransferase activity of UGGT2, whose specificity was shown to be quite similar to UGGT1, in terms of both glycan specificity and preferential recognition of proteins having non-native conformations. Finally, Sep15 was found to form the heterodimeric complex with both isoforms of UGGT and markedly enhanced its glucosyltransferase activity.

Localized Surface Plasmon Resonance Detection of Biological Toxins Using Cell Surface Oligosaccharides on Glyco Chips

We have detected biological toxins using localized surface plasmon resonance (LSPR) and synthetic glycosyl ceramides (β-lactoside, globosyl trisaccharide (Gb3), or GM1 pentasaccharide) attached to gold (Au) nanoparticles. The particle diameters ranged from 5-100 nm. The detection sensitivity for three toxins (ricin, Shiga toxin, and cholera toxin) was found to depend not only on the attached glycoside but also on the diameter of the Au nanoparticles. For the detection of ricin, the 20-nm β-lactoside-coated Au nanoparticle exhibited the highest LSPR response, whereas 40-nm Gb3- and GM1-coated Au nanoparticles gave the best results for Shiga toxin and cholera toxin, respectively. In addition, a blocking process on the nanoparticle surface greatly improved the detection sensitivity for cholera toxin. The LSPR system enabled us to detect ricin at 30 ng/mL, Shiga toxin at 10 ng/mL, and the cholera toxin at 20 ng/mL.

Combinatorial synthesis of oligosaccharide library of 2,6-dideoxysugars

Abstract Two combinatorial libraries composed of linear trisaccharides of 2,6-dideoxy- l -sugars have been constructed: All possible regioisomeric trisaccharides with 2-iodo and with 2-deoxy moieties were synthesized by an iodonium ion-catalyzed “stereoselective-yet-nonregioselective” glycosylation reaction.

Folding of Synthetic Homogeneous Glycoproteins in the Presence of a Glycoprotein Folding Sensor Enzyme

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.

Bisubstrate Analogues as Glycosyltransferase Inhibitors

Oligosaccharides in glycoconjugates such as glycoproteins and glycolipids play important roles in a variety of biological functions. Since glycosyltransferases are responsible for the biosynthesis of these oligosaccharides, inhibitors of glycosyltransferases are targets for drug discovery. Bisubstrate analogues, in which donor and acceptor analogue are covalently attached to each other, offer donors high affinity and acceptors high selectivity. In this review, we describe the design and synthesis of bisubstrate analogues of glycosyltransferases as well as their inhibitory potency hoping to inform the development of potent and selective inhibitors.

Chemical synthesis of erythropoietin glycoforms for insights into the relationship between glycosylation pattern and bioactivity

Chemical synthesis of homogeneous erythropoietins proved that their bioactivity in vivo is regulated by the glycosylation pattern. The role of sialyloligosaccharides on the surface of secreted glycoproteins is still unclear because of the difficulty in the preparation of sialylglycoproteins in a homogeneous form. We selected erythropoietin (EPO) as a target molecule and designed an efficient synthetic strategy for the chemical synthesis of a homogeneous form of five EPO glycoforms varying in glycosylation position and the number of human-type biantennary sialyloligosaccharides. A segment coupling strategy performed by native chemical ligation using six peptide segments including glycopeptides yielded homogeneous EPO glycopeptides, and folding experiments of these glycopeptides afforded the correctly folded EPO glycoforms. In an in vivo erythropoiesis assay in mice, all of the EPO glycoforms displayed biological activity, in particular the EPO bearing three sialyloligosaccharides, which exhibited the highest activity. Furthermore, we observed that the hydrophilicity and biological activity of the EPO glycoforms varied depending on the glycosylation pattern. This knowledge will pave the way for the development of homogeneous biologics by chemical synthesis.

Mannose‐BSA Conjugates: Comparison Between Commercially Available Linkers in Reactivity and Bioactivity

Mannosyl ethanolamine and BSA were conjugated together by their amino groups with various homobifunctional cross‐linker reagents: disuccinimidyl carbonate (DSC), disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), ethylene glycolbis(succinimidylsuccinate) (EGS), 1,5‐difluoro‐2,4‐dinitrobenzene (DFDNB), diethyl squarate (DES), and thiophosgene (TP). The resulting mannose-BSA conjugates were subjected to an enzyme‐linked lectin assay (ELLA)to investigate their affinity to concanvalin A (ConA). With these results, the seven linkers were evaluated on the basis of five criteria, i.e., cost, reactivity, sugar loading, homogeneity, and affinity to ConA. Thus, DSS, DFDNB, and DES seemed to have advantages over the other cross-linking reagents.