Reiko Sadamoto

Functional Neoglycopeptides: Synthesis and Characterization of a New Class of MUC1 Glycoprotein Models Having Core 2-Based O-Glycan and Complex-Type N-Glycan Chains

An efficient protocol for the construction of MUC1-related glycopeptide analogues having complex O-glycan and N-glycan chains was established by integrating chemical and enzymatic approaches on the functional polymer platforms. We demonstrated the feasibility of sortase A-mediated ligation between two glycopeptide segments by tagging with signal peptides, LPKTGLR and GG, at each C- or N-terminal position. Structural analysis of the macromolecular N,O-glycopeptides was performed by means of ESI-TOFMS (MS/MS) equipped with an electron-captured dissociation device. Immunological assay using MUC1 glycopeptides synthesized in this study revealed that N-glycosylation near the antigenic O-glycosylated PDTR motif did not disturb the interaction between the anti-MUC1 monoclonal antibody and this crucial O-glycopeptide moiety. NMR study indicated that the N-terminal immunodominant region [Ala-Pro-Asp-Thr(O-glycan)-Arg] forms an inverse gamma-turn-like structure, while the C-terminal region composed of N-glycopeptide and linker SrtA-peptide was proved to be an independently random structure. These results indicate that the bulky O- and N-glycan chains can function independently as disease-relevant epitopes and ligands for carbohydrate-binding proteins, when both are combined by an artificial intervening peptide having a possible effect of separating N- and C-terminal regions. The present strategy will greatly facilitate rapid synthesis of multiply functionalized complex neoglycopeptides as new types of convenient tools or models for the investigation of thhe structure-function relationship of various glycoproteins and development of novel class glycopeptide-based biopharmaceuticals, drug delivery systems, and biomedical materials.

Acceptor specificity and inhibition of the bacterial cell-wall glycosyltransferase MurG.

A continuous fluorescence coupled enzyme assay was developed to study the acceptor specificity of the glycosyltransferase MurG toward different lipid I analogues with various substituents replacing the undecaprenyl moiety. It was found that most lipid I analogues are accepted as substrates and, amongst these, the saturated C14 analogue exhibits the best activity. This substrate was used to evaluate the inhibition activity of such antibiotics as moenomycin, vancomycin, and two chlorobiphenyl vancomycin derivatives. A vancomycin derivative with a chlorobiphenyl moiety on the aglycon section was identified as a potent inhibitor of MurG.

Cell wall engineering of living bacteria through biosynthesis

Cell wall precursors that have been modified at their peptide moiety were incorporated into the living bacterial cell wall. Using chemically synthesized bacterial cell wall precursors, a variety of compounds could be attached to the bacterial surface. Escherichia coli took the modified precursors into the cell wall after EDTA treatment, whereas lactobacilli took the compounds more effectively without EDTA treatment. Microscopic observation showed that the incorporated ketone moiety retained its reactivity. On the basis of this strategy, any compound can be displayed on the bacterial surface. This strategy for bacterial cell surface engineering will open the door for new technologies and therapies utilizing bacteria.

Bacterial surface engineering utilizing glucosamine phosphate derivatives as cell wall precursor surrogates.

Surface display on bacteria has attracted much attention due to the potential applications in peptide library screening, and in the syntheses of various bioadsorbants, biosensors, and oral vaccines. Therefore, the cell-surface engineering of bacteria has been an important theme in the field of industry and in particular biotechnology. Several approaches to cell surface engineering have been explored thus far. Genetic techniques have been used to express target proteins as conjugates with their native surface proteins. However, the approaches are limited to the display only of proteins and peptides. In contrast to the genetic approaches, a chemical approach based on the metabolic pathway has been utilized to display a variety of molecules. Metabolic labeling of cell-surface biomolecules with a non-native chemical tag allows for the covalent attachment of various synthetic epitopes. Bertozzi et al. originally proved that unnatural carbohydrate substrates could be incorporated into an oligosaccharide to introduce novel chemical reactivity to mammalian cell surfaces. In the case of bacteria, Tirrell and co-workers successfully incorporated an azide group, using an azido amino acid as a methionine surrogate, into an outer membrane protein of Escherichia coli in which additional methionine residues were engineered by site-directed mutagenesis. We previously developed an approach to display various kinds of molecules on the bacterial cell surface using chemically synthesized cell-wall (peptidoglycan) precursors. Since the composition of peptidoglycan is strikingly similar between bacterial strains, our approach is applicable to a variety of bacteria. In this method, chemically synthesized cell-wall precursors, that is, UDP-MurNAc pentapeptide derivatives, are incorporated into the cell wall through a biosynthetic pathway; this methods does not depend on genetic modifications, thereby presenting a range of potential target moieties, such as oligosaccharides, beyond that of conventional protein display. However, as the synthesis of the UDP-MurNAc pentapeptide derivative is difficult and timeconsuming, the large-scale application of this system is problematic. To overcome these problems, we focused on N-acetylglucosamine-1-phosphate (GlcNAc-1-phosphate), which is an early, simple intermediate in the peptidoglycan biosynthetic route (Figure 1), and herein present our results. In bacteria, GlcNAc-1-phosphate can be an intermediate of both the MurNAc-pentapeptide and GlcNAc components of peptidoglycans (see Figure 1). In bacterial cell-wall biosynthesis, GlcNAc-1-phosphate is formed from glucosamine-1-phosphate, whereas in mammalian cells, GlcNAc is directly converted to the phosphorylated form via salvage pathways. Therefore, we chose GlcNAc-1-phosphate, and not GlcNAc, as the platform precursor for the introduction of non-native reactive groups into the bacterial cell wall, and designed compound 1, in which a ketone group was introduced into the N-acetyl group, as a bacterial cell wall pre[a] Dr. R. Sadamoto Present address: Ochadai Academic Production, The Glycoscience Institute Ochanomizu University, 2-1-1, Otsuka, Bunkyo-ku Tokyo 112-8610 (Japan) Fax: (+81) 3-5978-2581 E-mail : sadamoto.reiko@ocha.ac.jp [b] Dr. R. Sadamoto, Dr. S. Koshida Shionogi Laboratory of Biomolecular Chemistry Graduate School of Advanced Life Science, Hokkaido University Kita-21, Nishi-11, Sapporo 001-0021 (Japan) [c] T. Matsubayashi, M. Shimizu, T. Ueda, Prof. S.-I. Nishimura Laboratory for Bio-Macromolecular Chemistry Graduate School of Advanced Life Science, Hokkaido University Kita-21, Nishi-11, Sapporo 001-0021 (Japan) [d] Prof. T. Koda Laboratory of Embryonic and Genetic Engineering Graduate School of Advanced Life Science, Hokkaido University Kita-21, Nishi-11, Sapporo 001-0021 (Japan) [e] Prof. S.-I. Nishimura Drug-Seeds Discovery Research Laboratory National Institute of Advanced Industrial Science and Technology (AIST) Sapporo 062-8517 (Japan) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200801734.

Synthesis of Photopolymerizable Glycolipids and their Application as Scaffolds to Immobilize Proteins with a Micron-Sized Pattern

Photopolymerizable glycolipids incorporating ceramide- or amido-type linkers and able to form stable monolayers were efficiently synthesized by chemical and enzymatic methods. Glycolipid polymer films served as platforms for the immobilization of proteins through specific carbohydrate–protein interactions at the air–water interface. Carbohydrate-binding proteins deposited on the glycolipid film were observed by atomic force microscopy, which showed varying submicron-sized protein patterns such as dendrites, dots, and networks, depending on the lipid structure, membrane preparation process, and sugar density of the membrane. Surface plasmon resonance measurement confirmed that the subunit-type lectins immobilized on the glycolipid membranes exhibited the ability to interact specifically with carbohydrate ligands by using unoccupied binding sites.

Free volumes in nematic and smectic liquid-crystalline polymers probed by positron annihilation

Free volumes in thermotropic side-chain liquid-crystalline polymers were probed by positron annihilation technique. Lifetime spectra of positrons were measured in the temperature range between 130 and −60°C in cooling. For a nematic liquid-crystalline polymer (polyacrylate), the lifetime of ortho-positronium (τ3) was decreased with decreasing temperature above the glass transition temperature (Tg, 21°C) with larger temperature coefficient than that below Tg. The intensity of ortho-positronium (I3) was constant above Tg. These facts mean that the size of the free-volume holes decreased with the decreasing the temperature but the concentration was almost constant in nematic phase. For a smectic liquid-crystalline polymer (poly(p-methylstyrene) derivative), a discontinuous decrease in the value of τ3 and that of I3 were observed at 107°C, which was the transition temperature from smectic to crystalline phase. Such discontinuous changes were not observed for the polyacrylate specimen. This difference was considered to be attributed to the higher-ordered structure of the smectic phase.