Shunji Natsuka

Structure-function analysis of human IL-6 receptor : dissociation of amino acid residues required for IL-6-binding and for IL-6 signal transduction through gp130

Here, we report the analysis of the structure‐function relationship of the extracellular region of human interleukin 6 receptor (IL‐6R). Upon binding of IL‐6, IL‐6R becomes associated extracellularly with a non‐IL‐6‐binding but signal transducing molecule, gp130, and the IL‐6 signal is generated. In this region, the cytokine receptor family domain, but not the immunoglobulin‐like domain, was responsible both for IL‐6 binding and for signal transduction through gp130. Because a soluble, extracellular portion of IL‐6R (sIL‐6R) could bind IL‐6 and mediate IL‐6 functions through gp130, amino acid substitutions were introduced into sIL‐6R by site‐directed mutagenesis. The results, together with the previously proposed tertiary structure model, suggested that the amino acid residues critical for IL‐6 binding have a tendency to be distributed to the hinge region between the two ‘barrel’‐like fibronectin type III modules and to the same side of these two ‘barrels’. Amino acid residues, of which substitutions barely affected the IL‐6‐binding but did abolish the IL‐6 signalling capability of sIL‐6R, were identified and found to be located mainly in the membrane proximal half of the second barrel. sIL‐6R mutants carrying such substitutions lacked the capacity to associate with gp130 in the presence of IL‐6.

Identification method for twelve oligomannose-type sugar chains thought to be processing intermediates of glycoproteins

A mixture of the pyridylamino (PA) derivatives of 12 oligomannose-type sugar chains was fractionated into five fractions (mannose5N-acetylglucosamine2-PA approximately mannose9N-acetylglucosamine2-PA) by size-fractionation HPLC with a MicroPak AX-5 column. Each fraction thus obtained was then analyzed by reversed-phase HPLC with a Cosmosil 5C18-P column. In this way, the 12 PA-oligomannose-type sugar chains were completely separated from each other. The method was used to identify the structures of oligomannose-type sugar chains of human C3, the third component of human complement.

Fluorescence method for the structural analysis of oligomannose-type sugar chains by partial acetolysis☆

Fluorescence labeling was used in the analysis of partial acetolysis products of oligomannose-type sugar chains with five to nine mannose residues. The principle of the method was the pyridylamination of fragments obtained by the partial acetolysis of pyridylamino sugar chains and the identification of the fragments with an HPLC apparatus equipped with a fluorescence spectrophotometer. The method was tested by analysis of eight oligomannose-type sugar chains with known chemical structures and was found to be effective for analysis of branching structures with samples of 0.5 nmol.

CO-OVEREXPRESSION OF FOLDING MODULATORS IMPROVES THE SOLUBILITY OF THE RECOMBINANT GUINEA PIG LIVER TRANSGLUTAMINASE EXPRESSED IN ESCHERICHIA COLI

ABSTRACT Transglutaminases (EC 2.3.2.13) catalyze the formation of ε-(γ-glutamyl)lysine cross-links and the substitution of primary amines for the γ-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°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.

Structural analysis of N-glycans of the planarian Dugesia japonica

To investigate the relationship between phylogeny and glycan structures, we analyzed the structure of planarian N‐glycans. The planarian Dugesia japonica, a member of the flatworm family, is a lower metazoan. N‐glycans were prepared from whole worms by hydrazinolysis, followed by tagging with the fluorophore 2‐aminopyridine at their reducing end. The labeled N‐glycans were purified, and separated by three HPLC steps. By comparison with standard pyridylaminated N‐glycans, it was shown that the N‐glycans of planarian include high mannose‐type and pauci‐mannose‐type glycans. However, many of the major N‐glycans from planarians have novel structures, as their elution positions did not match those of the standard glycans. The results of mass spectrometry and sugar component analyses indicated that these glycans include methyl mannoses, and that the most probable linkage was 3‐O‐methylation. Furthermore, the methyl residues on the most abundant glycan may be attached to the non‐reducing‐end mannose, as the glycans were resistant to α‐mannosidase digestion. These results indicate that methylated high‐mannose‐type glycans are the most abundant structure in planarians.

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.

The och1 Mutant of Schizosaccharomyces pombe Produces Galactosylated Core Structures of N-Linked Oligosaccharides

Unlike the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe synthesizes large outer chains on the N-linked oligosaccharides that consist mainly of D-Gal and D-Man residues. The fission yeast och1 + gene product has α1,6-mannosyltransferase activity, and Och1p is the key enzyme in the initiation of outer chain elongation. Although the in vitro substrate specificity of S. pombe Och1p has been reported (Yoko-o et al., FEBS Lett., 489, 75–80 (2001)), the structure of the N-linked oligosaccharides of och1Δ cells has not been investigated. In this study, we report a structural analysis of S. pombe N-linked oligosaccharides. Lectin blot analysis indicated that galactose residues were attached to the cell surface glycoproteins of the och1Δ cells. We conducted a structural analysis of pyridylaminated N-linked oligosaccharides prepared from galactomannoproteins by HPLC and 1H NMR. These analyses revealed that the N-linked oligosaccharides of the och1Δ cells displayed heterogeneity in the glycan consisting of Hex11–15GlcNAc2. The structural heterogeneity arose mainly from the addition of α1,2- and α1,3-Gal residues to the Man9GlcNAc2 core structure.

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 HPLC’s. The comparison of the elution profiles of HPLC’s 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.

N-Glycosylation patterns of hemolymph glycoproteins from Biomphalaria glabrata strains expressing different susceptibility to Schistosoma mansoni infection

Comparative analyses of the N-glycosylation pattern of hemolymph glycoproteins from Biomphalaria glabrata strains Puerto Rico (BgPR) and Salvador (BgBS-90), differing in their susceptibility towards Schistosoma mansoni infection, were performed by Western blotting, enzyme-linked immunosorbent assays, two-dimensional high-performance liquid chromatography and mass spectrometry. Obtained data demonstrated an enhanced expression of serologically cross-reacting, fucosylated carbohydrate epitopes by the highly susceptible BgPR-strain in comparison to the resistant BgBS-90-strain. In particular, glycoproteins of BgPR snails exhibited larger amounts of glycans with (β1-2)-linked xylose or terminal Fuc(α1-3)GalNAc(β1-4)[±Fuc(α1-3)]GlcNAc(β1-)-units which are known to mediate cross-reactivity with schistosomal glycoconjugates. This finding could be corroborated by immunohistochemical studies showing again an enhanced expression of such carbohydrate epitopes in BgPR tissue. Hence, our results provide evidence for a correlation of B. glabrata susceptibility towards S. mansoni infection and the expression of carbohydrate determinants shared by the parasite and its intermediate host.

Structure determination of a sulfated N-glycans, candidate for a precursor of the selectin ligand in bovine lung

To clarify the structure of non-sialic acid anionic residue on N-glycans in the mammalian tissues, we have isolated sialidase-resistant anionic residue on N-glycans from bovine lung. Analyses by partial acid hydrolysis and glycosidase digestions combined with a two-dimensional HPLC mapping method revealed that the major sialidase-resistant anionic N-glycan had a fucosylbianntenary core structure. The anionic residue was identified as a sulfate ester by methanolysis, anion-exchange chromatography, and mass spectrometry. The linkage position of the sulfate ester was the 6-position of the GlcNAc residue on the Manα1-6 branch. This conclusion was based on the results of glycosidase digestions followed by two-dimensional HPLC mapping. Furthermore, the disialylated form of this sulfated glycan was dominant, and no asialo form was detected. The structure of the major anionic N-glycan prepared from bovine lung and having a sulfate was proposed to be the pyridylamino derivative of Siaα2-3Gαlβ1-4(HSO3-6)GlcNAcβ1-2Manα1-6(Siaα2-3Galβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc.