A polymer-type water-soluble peptidoglycan exhibited both Toll-like receptor 2- and NOD2-agonistic activities, resulting in synergistic activation of human monocytic cells

Bacterial peptidoglycan (PGN) has been reported to be sensed by cell-surface Toll-like receptor (TLR)2. On the other hand, intracellular NOD-like receptors recognize PGN partial structures: NOD1 and NOD2 recognize the peptide moiety containing diaminopimelic acid, and the muramyldipeptide (MDP) moiety, respectively. In this study, we examined in human monocytic THP-1 cells the pro-inflammatory cytokine-inducing abilities of PGNs and their fragments enzymatically prepared from Staphylococcus epidermidis ATCC 155: a polymer-type water-soluble PGN possessing an intact glycan chain (SEPS) and a monomer-type PGN (SEPS-M). The water-soluble PGN polymer, SEPS, exhibited considerably stronger activities to induce pro-inflammatory cytokines than parent PGNs and the PGN monomer, SEPS-M. Short interference RNA targeting TLR2 and NOD2 markedly reduced the activities of SEPS. In the same experiments, the activities of PGNs were mainly reduced in TLR2-silenced cells, whereas the activities of SEPS-M as well as a synthetic MDP were markedly reduced in NOD2-silenced cells. Furthermore, the PGNs and a reference PGN from Staphylococcus aureus in combination with MDP synergistically induced interleukin-8 in THP-1 cells. These findings strongly suggested that a polymer-type water-soluble PGN fragment, SEPS, exhibits both TLR2-and NOD2-agonistic activities, which induced the synergistic activation of human monocytic cells.     

Structural analysis of N-linked glycans in Caenorhabditis elegans

Caenorhabditis elegans is an excellent model for morphogenetic research. However, little information is available on the structure of cell-surface glycans in C. elegans, although several lines of evidence have suggested a role for these glycans in cell-cell interactions during development. In this study, we analyzed N-glycan structures. Oligosaccharides liberated by hydrazinolysis from a total membrane fraction were labeled by pyridylamination, and around 90% of the N-glycans were detected as neutral oligosaccharides. The most dominant structure was Man(alpha)1-6(Man(alpha)1-3)Man(beta)1-4GlcNAc(beta)1-4GlcNAc, which is commonly found in insects. Branching structures of major oligomannose-type glycans were the same as those found in mammals. Structures that had a core fucose or non-reducing end N-acetylglucosamine were also identified, but ordinary complex-type glycans with N-acetyllactosamine were not detected as major components.     

Expression of complex-type N-glycans in developmental periods of zebrafish embryo

As a first step to elucidate a role of N-glycans in development of vertebrates, we analyzed structures of the glycans expressed in early stages of zebrafish embryo. N-glycans were prepared from zebrafish embryos at several developmental stages followed by tagging with a fluorophore, 2-aminopyridine. The labeled glycans were analyzed by two modes of HPLCs. The comparison of the elution profiles of HPLCs unveil the change of the oligosaccharide structure during the development. These peaks were merely detected during 4–7 h after fertilization, however, increased from 12 h, and at 15 h a fairly amount of them was appeared. Structure analysis revealed that they were bianntenary complex-type N-glycans with or without fucose and/or bisecting N-acetylglucosamine residues. These results suggest that the complex-type N-glycans are concerned in some developmental event from segmentation period downward in zebrafish. Published in 2005.     

Method for purification of fluorescence-labeled oligosaccharides by pyridylamination

We developed a convenient method for purification of PA-oligosaccharides to remove contaminants originating from natural fluorescent materials, and excess reagents as well as by-products of tagging reactions in glycan analysis. The method, using a C18-cartridge, is simple and powerful to remove them. Several examples of experiments that showed the usefulness of this purification method are described in this report.     

Identification of an endo-beta-N-acetylglucosaminidase gene in Caenorhabditis elegans and its expression in Escherichia coli

We report the identification, molecular cloning, and characterization of an endo-beta-N-acetylglucosaminidase from the nematode Caenorhabditis elegans. A search of the C. elegans genome database revealed the existence of a gene exhibiting 34% identity to Mucor hiemalis (a fungus) endo-beta-N-acetylglucosaminidase (Endo-M). Actually, the C. elegans extract contained endo-beta-N-acetylglucosaminidase activity. The putative cDNA for the C. elegans endo-beta-N-acetylglucosaminidase (Endo-CE) was amplified by polymerase chain reaction from the Uni-ZAP XR library, cloned, and sequenced. The recombinant Endo-CE expressed in Escherichia coli exhibited substrate specificity mainly for high-mannose type oligosaccharides. Man(8)GlcNAc(2) was the best substrate for Endo-CE, and Man(3)GlcNAc(2) was also hydrolyzed. Biantennary complex type oligosaccharides were poor substrates, and triantennary complex substrates were not hydrolyzed. Its substrate specificity was similar to those of Endo-M and endo-beta-N-acetylglucosaminidase from hen oviduct. Endo-CE was confirmed to exhibit transglycosylation activity, as seen for some microbial endo-beta-N-acetylglucosaminidases. This is the first report of the molecular cloning of an endo-beta-N-acetylglucosaminidase gene from a multicellular organism, which shows the possibility of using this well-characterized nematode as a model system for elucidating the role of this enzyme.     

beta1-4Galactosyltransferase activity of mouse brain as revealed by analysis of brain-specific complex-type N-linked sugar chains

We previously reported two brain-specific agalactobiantennary N-linked sugar chains with bisecting GlcNAc and alpha1-6Fuc residues, (GlcNAcbeta1-2)(0)(or)(1)Manalpha1-3(GlcNAcbeta1-2M analpha1-6)(GlcNA cbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)Glc NAc [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. 114, 334-338]. Here, the reason for the absence of Gal on the sugar chains was analyzed through the detection of other complex type sugar chains. Analysis of N-linked sugar chains revealed the absence of Sia-Gal and Gal on the GlcNAc residues of brain-specific agalactobiantennary N-linked sugar chains. We therefore investigated the substrate specificity of galactosyltransferase activities in brain using pyridylamino derivatives of agalactobiantennary sugar chains with structural variations in the bisecting GlcNAc and alpha1-6Fuc residues as acceptor substrates. While the beta1-4galactosyltransferases in liver and kidney could utilize all four oligosaccharides as substrates, the beta1-4galactosyltransferase(s) in brain could not utilize the agalactobiantennary sugar chain with both bisecting GlcNAc and Fuc residues, but could utilize the other three acceptors. Similar results were obtained using glycopeptides with agalactobiantennary sugar chains and bisecting GlcNAc and alpha1-6Fuc residues as substrates. The beta1-4galactosyltransferase activity of adult mouse brain thus appears to be responsible for producing the brain-specific sugar chains and to be different from beta1-4galactosyltransferase-I. The agalactobiantennary sugar chain with bisecting GlcNAc and alpha1-6Fuc residues acts as an inhibitor against "brain type" beta1-4galactosyltransferase with a K(i) value of 0.29 mM.     

Co-overexpression of folding modulators improves the solubility of the recombinant guinea pig liver transglutaminase expressed in Escherichia coli

Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of primary amines for the gamma-carboxamide groups of protein bound glutamine residues, and are involved in many biological phenomena. Transglutaminase reactions are also applicable in applied enzymology. Here, we established an expression system of recombinant mammalian tissue-type transglutaminase with high productivity. Overexpression of guinea pig liver transglutaminase in Escherichia coli, using a plasmid pET21-d, mostly resulted in the accumulation of insoluble and inactive enzyme protein. By the expression culture at lower temperatures (25 and 18 degrees C), however, a fraction of the soluble and active enzyme protein slightly increased. Co-overexpression of a molecular chaperone system (DnaK-DnaJ-GrpE) and/or a folding catalyst (trigger factor) improved the solubility of the recombinant enzyme produced in E. coli cells. The specific activity, the affinity to the amine substrate, and the sensitivity to the calcium activation and GTP inhibition of the purified soluble recombinant enzyme were lower than those of the natural liver enzyme. These results indicated that co-overexpression of folding modulators tested improved the solubility of the overproduced recombinant mammalian tissue-type transglutaminase, but the catalytic properties of the soluble recombinant enzyme were not exactly the same as those of the natural enzyme.     

Augmentation of haptoglobin production in Hep3B cell line by a nuclear factor NF-IL6.

The nuclear factor NF-IL6 had been suggested to be responsible for the IL-6-mediated induction of several acute-phase proteins. To obtain evidence for the involvement of NF-IL6 in the induction of acute-phase proteins, we introduced the NF-IL6 gene and its truncated mutant (delNFIL6) gene into a hepatoma cell line Hep3B. Then, we examined the effect of the overproduced NF-IL6 and delNFIL6 on the expression of haptoglobin, fibrinogen and albumin. As a result, basal production as well as induction of haptoglobin by IL-6 were augmented by the expression of NF-IL6, whereas delNFIL6 blocked the production of haptoglobin, fibrinogen and albumin.