Highly oriented recombinant glycosyltransferases: site-specific immobilization of unstable membrane proteins by using Staphylococcus aureus sortase A.

Recombinant glycosyltransferases are potential biocatalysts for the construction of a compound library of oligosaccharides, glycosphingolipids, glycopeptides, and various artificial glycoconjugates on the basis of combined chemical and enzymatic synthetic procedures. The structurally defined glycan-related compound library is a key resource both in the basic studies of their functional roles in various biological processes and in the discovery research of new diagnostic biomarkers and therapeutic reagents. Therefore, it is clear that the immobilization of extremely unstable membrane-bound glycosyltransferases on some suitable supporting materials should enhance the operational stability and activity of recombinant enzymes and makes facile separation of products and recycling use of enzymes possible.

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.

Molecular design of glycoprotein mimetics: glycoblotting by engineered proteins with an oxylamino-functionalized amino acid residue.

The general and efficient method for the site-directed glycosylation of proteins is a key step in order to understand the biological importance of the carbohydrate chains of proteins and to control functional roles of the engineered glycoproteins in terms of the development of improved glycoprotein therapeutics. We have developed a novel method for site-directed glycosylation of proteins based on chemoselective blotting of common reducing sugars by genetically encoded proteins. The oxylamino-functionalized L-homoserine residues, 2-amino-4-O-(N-methylaminooxy) butanoic acid and 2-amino-4-aminooxy butanoic acid, were efficiently incorporated into proteins by using the four-base codon/anticodon pair strategy in Escherichia coli in vitro translation.

Glycoinsulins: dendritic sialyloligosaccharide-displaying insulins showing a prolonged blood-sugar-lowering activity.

Mono-, di-, and trisialyloligosaccharides were introduced to mutant insulins through enzymatic reactions. Sugar chains were sialylated by alpha2,6-sialyltransferase (alpha2,6-SiaT) via an accessible glutamine residue at the N-terminus of the B-chain attached by transglutaminase (TGase). Sia2,6-di-LacNAc-Ins(B-F1Q) and Sia2,6-tri-LacNAc-Ins(B-F1Q), displaying two and three sialyl-N-acetyllactosamines, respectively, were administered to hyperglycemic mice.

Chemo-enzymatic synthesis of fluorinated 2-N-acetamidosugar nucleotides using UDP-GlcNAc pyrophosphorylase.

Two non-natural fluorinated 2-N-acetamidosugar nucleotides, uridine 5'-diphosphate (UDP) 2-acetamido-2,4-dideoxy-4-fluoro-alpha-D-glucopyranose (UDP-4-FGlcNAc) 1 and its galacto isomer (UDP-4-FGalNAc) 2, were enzymatically constructed by treating chemically synthesized fluorinated 2-N-acetamidosugar 1-phosphates as the donor with UDP 2-acetamido-2-deoxy-alpha-D-glucopyranose pyrophosphorylase in the presence of uridine 5'-triphosphate (UTP).

Synthesis of 4-fluorinated UDP-MurNAc pentapeptide as an inhibitor of bacterial growth.

4-Fluorinated UDP-MurNAc pentapeptide, 2, has been synthesized. In our previous study, UDP-MurNAc pentapeptide analogue 1 was found to be incorporated into the bacterial cell wall through biosynthesis. Compound 2 showed growth-inhibition activity against Gram-positive bacteria when it was added to growth media at 0.

Control of bacteria adhesion by cell-wall engineering.

UDP-MurNAc-pentapeptide derivative bacterial cell-wall precursors were synthesized as effective tools for surface display on living bacteria. Lactobacilli were incubated in the ketone-modified precursor-containing medium, and the ketone moiety was displayed on the bacterial surface through cell-wall biosynthesis. Oligomannose was coupled with the ketone moiety on the bacterial surface via a aminooxyl linker, thereby displaying this oligosaccharide on the surface of the bacteria.

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.

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.

Cell-wall engineering of living bacteria.

The cell walls of living bacteria were chemically modified by adding cell-wall precursors. As the precursors to be incorporated into the cell wall, UDP-MurNAc pentapeptide, lipid I, and lipid II derivatives were synthesized. The aimed compounds were attached to the amine residue of lysine at the pentapeptide moiety.