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F Inagaki

Publications and source records attributed to F Inagaki.

At least 37 records · Page 2Linked to original sources
PubMed1999

Analysis of human IgE epitope of Der f 2 with anti-Der f 2 mouse monoclonal antibodies.

Der f 2 is one of the major mite allergens recognized by human IgE antibodies of allergic patients. Using five anti-Der f 2 mouse monoclonal antibodies, human IgE epitopes of Der f 2 were analyzed. Among them, two monoclonal antibodies 15E11 and 13A4 inhibited the binding between Der f 2 and human IgE antibodies. To determine major IgE epitopes of Der f 2, epitopes for the monoclonal IgG antibodies were analyzed using 43 single site Der f 2 mutants constructed by site-directed mutagenesis. Binding ability of 13A4 and 15E11 was decreased by the amino acid replacement around the C-terminus, and around 73rd, respectively. These results suggest that the C-terminal portion and the central portion around 73rd of Der f 2 were recognized by human IgE antibodies as major epitopes. The location of the putative IgE epitopes on 3-D structure of Der f 2 is also discussed.

Allergens↗
PubMed1999

Solution structure of human acidic fibroblast growth factor and interaction with heparin-derived hexasaccharide.

Fibroblast growth factors (FGFs) bind to extracellular matrices, especially heparin-like carbohydrates of heparan-sulfate proteoglycans which stabilize FGFs to protect against inactivation by heat, acid, proteolysis and oxidation. Moreover, binding of FGFs to cell surface proteoglycans promotes to form oligomers, which is essential for receptor oligomerization and activation. In the present study, we determined the solution structure of acidic FGF using a series of triple resonance multi-dimensional NMR experiments and simulated annealing calculations. Furthermore, we prepared the sample complexed with a heparin-derived hexasaccharide which is a minimum unit for aFGF binding. From the chemical shift differences between free aFGF and aFGF-heparin complex, we concluded that the major heparin binding site was located on the regions 110-131 and 17-21. The binding sites are quite similar to those observed for bFGF-heparin hexasaccharide complex, showing that both FGFs recognize heparin-oligosaccharides in a similar manner.

Amino Acid Sequence↗
PubMed1999

Solution structure of the SH2 domain of Grb2/Ash complexed with EGF receptor-derived phosphotyrosine-containing peptide.

1H, 13C, and 15N NMR resonances of the SH2 domain of Grb2/Ash in both the free form and the form complexed with a phosphotyrosine-containing peptide derived from the EGF receptor were assigned by analysis of multi-dimensional, double- and triple-resonance NMR experiments. From the chemical shift changes of individual residues upon peptide binding, the binding site for the peptide was mapped on the structure of Grb2/Ash SH2. The peptide was not recognized by the groove formed by the BG and EF loops, suggesting that the EGFR peptide does not bind to Grb2/Ash SH2 in an extended conformation. This was supported by analysis of the binding affinity of mutants where residues on the BG and EF loops were changed to alanine. The present results are consistent with the recently reported structures of Grb2/Ash SH2 complexed with BCR-Abl and Shc-derived phosphotyrosine containing peptides, where the peptide forms a turn conformation. This shows that the specific conformation of the phosphotyrosine-containing sequence is required for the SH2 binding responsible for downstream signaling.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Disulfide bond structure of human epidermal growth factor receptor.

The extracellular domain of the human epidermal growth factor receptor (sEGFR) consists of 621 amino acid residues, including 50 cysteines. The connections of the 25 disulfide bonds in the recombinant sEGFR protein, obtained from Chinese hamster ovary cells, have been determined using N-terminal sequencing and matrix-assisted laser desorption/ionization mass spectroscopy. We identified a basic repeat of eight cysteines with a 1-3, 2-4, 5-6, and 7-8 disulfide pairing pattern in the two cysteine-rich regions of sEGFR. By comparison to other cysteine-rich motifs, it was concluded that the cysteine-rich repeat of sEGFR belongs to the laminin-type EGR-like (LE) structural motif. Three-dimensional structure models of the two cysteine-rich regions have been built, based on the three-dimensional structures of the LE domains from the laminin gamma1 chain and secondary structure predictions for the EGF receptor.

Amino Acid Sequence↗
PubMed1998

Ganglioside composition of GH3 cells: enhancement of fucoganglioside expression by estradiol, epidermal growth factor and insulin.

The GH3 cell line, a bipotential cell line secreting both prolactin (PRL) and growth hormone (GH), is a useful model for investigating GH/PRL cell lineage differentiation and anterior pituitary adenoma formation. In this study, we investigated the ganglioside composition of GH3 cells and identified two fucogangliosides as the major gangliosides expressed by these cells. Analyses by DEAE-Sephadex A-25 and thin-layer chromatography (TLC) revealed that the GH3 cells contained two major gangliosides, designated FG1 and FG2, respectively. Their structures were identified by fast atom bombardment mass spectrometry and proton nuclear magnetic resonance spectrometry: FG1 is IV2FUc alpha,II3NeuAc-GgOse4Cer and FG2 is IV2FUc alpha,IV3Gal alpha,II3NeuAc-GgOse4Cer. Expression of these fucogangliosides was enhanced by chronic treatment with 17 beta-estradiol (1 nM), epidermal growth factor (10 nM) and insulin (300 nM), which induced differentiation of GH3 cells to normal PRL-secreting cells. Interestingly, immunocytochemistry and flow cytometry revealed that the increased expression of these gangliosides reflected a quantitative change inside the cells but not on the cell surface. These results suggest that the intracellular distribution of fucogangliosides is closely related to the differentiation of GH3 cells.

Animals↗
PubMed1998

Solution structure of Der f 2, the major mite allergen for atopic diseases.

House dust mites cause heavy atopic diseases such as asthma and dermatitis. Among allergens from Dermatophagoides farinae, Der f 2 shows the highest positive rate for atopic patients, but its biological function in mites has been perfectly unknown, as well as the functions of its homologs in human and other animals. We have determined the tertiary structure of Der f 2 by multidimensional nuclear magnetic resonance spectroscopy. Der f 2 was found to be a single-domain protein of immunoglobulin fold, and its structure was the most similar to those of the two regulatory domains of transglutaminase. This fact, binding to the bacterial surface, and other small pieces of information hinted that Der f 2 is related to the innate antibacterial defense system in mites. The immunoglobulin E epitopes are also discussed on the basis of the tertiary structure.

Allergens↗
PubMed1998

Clusters of basic amino acids in midkine: roles in neurite-promoting activity and plasminogen activator-enhancing activity.

The removal of N-terminally located clusters of basic amino acids (N-tail) or C-terminally located clusters of basic amino acids (C-tail) from the midkine (MK) molecule severely reduced its neurite-promoting activity. However, experiments involving chemically synthesized MK derivatives revealed that the roles of the N-tail and C-tail were mostly indirect ones, i.e. they probably maintain the steric arrangements of the N-terminal and C-terminal halves. In particular, the C-domain, which is the C-terminal half devoid of the C-tail, retained considerable neurite-promoting activity when it was uniformly coated on a dish. The removal of the N-tail or C-tail also reduced the enhancing activity of plasminogen activator (PA) in aortic endothelial cells, although the effect was lower. There are two heparin-binding sites in the C-domain, Clusters I and II. A mutation in Cluster I [R78-->Q] affected the PA-enhancing activity only slightly, and a mutation in Cluster II [K83K84-->QQ] abolished the activity, while both mutations are known to reduce the neurite-promoting activity moderately. Therefore, the two heparin-binding sites in the C-domain play different roles in these two activities. Indeed, heparin exhibited different effects on these two activities. We also observed that intact MK was required for ordered neurite-promotion along the path of MK; one possible interpretation of this is that the N-terminal half is necessary for the stability of the molecule. Furthermore, K76 and K99 were found to be required for the secretion of MK; i.e. mutants in which one of these K residues was changed to Q were produced in the host cells, but not found in the medium.

Amino Acid Substitution↗
PubMed1997

Solution structure of midkine, a new heparin-binding growth factor.

Midkine (MK) is a 13 kDa heparin-binding polypeptide which enhances neurite outgrowth, neuronal cell survival and plasminogen activator activity. MK is structurally divided into two domains, and most of the biological activities are located on the C-terminal domain. The solution structures of the two domains were determined by NMR. Both domains consist of three antiparallel beta-strands, but the C-terminal domain has a long flexible hairpin loop where a heparin-binding consensus sequence is located. Basic residues on the beta-sheet of the C-terminal domain form another heparin-binding site. Measurement of NMR signals in the presence of a heparin oligosaccharides verified that multiple amino acids in the two sites participated in heparin binding. The MK dimer has been shown to be the active form, giving signals to endothelial cells and probably to neuronal cells. We present a head-to-head dimer model of MK. The model was supported by the results of cross-linking experiments using transglutaminase. The dimer has a fused heparin-binding site at the dimer interface of the C-terminal domain, and the heparin-binding sites on MK fit the sulfate group clusters on heparin. These features are consistent with the proposed stronger heparin-binding activity and biological activity of the dimer.

Amino Acid Sequence↗
PubMed1997

Identification of heparin-binding sites in midkine and their role in neurite-promotion.

Midkine (MK) is a heparin-binding growth factor, which promotes neurite outgrowth in embryonic neurons and enhances their survival. The three dimensional structure of MK clarified by NMR spectroscopy indicates that several basic amino acids are exposed on the surface of the C-terminal half domain, which retains heparin-binding and neurite-promoting activity. We performed site-directed mutagenesis of these amino acids, and found that mutation of arginine78 reduced the heparin-binding activity. Mutation of either lysine83 or lysine84 scarcely affected heparin-binding activity, while the double mutant involving both lysine residues showed reduction in the activity. Neurite-promoting activity of mutant MKs always correlated with their heparin-binding activity, illustrating the close relationship of the two activities. Thus, the present result verifies the occurrence of two distinct heparin-binding sites involved in neurite-promoting activity of MK molecule.

Animals↗
PubMed1997

Solution structure of ferredoxin from the thermophilic cyanobacterium Synechococcus elongatus and its thermostability.

The three-dimensional structure of ferredoxin, purified from the thermophilic cyanobacterium Synechococcus elongatus, was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance. In addition to the 946 distance constraints from nuclear Overhauser effect connectivities, we added 241 distance constraints derived from the crystal structure of Spirulina platensis ferredoxin to the 19 residues close to the [2Fe-2S] iron-sulfur center, where crosspeaks disappeared due to paramagnetic effects. The atomic root-mean-square difference of the ten converged structures from the mean structure was 0.61(+/-0.12) A for backbone atoms (N, C(alpha), C'). The main-chain structure was almost the same as the crystal structures of other mesophile ferredoxins, but comparison of the side-chain structures revealed an extension of the hydrophobic core, a unique hydrophobic patch on the surface of the large beta-sheet, and two unique charge networks in this thermostable ferredoxin structure, some of which might contribute to thermostability.

Amino Acid Sequence↗
PubMed1997

Conformation of an Shc-derived phosphotyrosine-containing peptide complexed with the Grb2 SH2 domain.

We have determined the structure of an Shc-derived phosphotyrosine-containing peptide complexed with Grb2 SH2 based on intra- and intermolecular NOE correlations observed by a series of isotope-filtered NMR experiments using a PFG z-filter. In contrast to an extended conformation of phosphotyrosine-containing peptides bound to Src, Syp and PLC gamma SH2s, the Shc-derived peptide formed a turn at the +1 and +2 positions next to the phosphotyrosine residue. Trp121, located at the EF1 site of Grb2 SH2, blocked the peptide binding in an extended conformation. The present study confirms that each phosphotyrosine-containing peptide binds to the cognate SH2 with a specific conformation, which gives the structural basis for the binding specificity between SH2s and target proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Ligand-binding enhances the affinity of dimerization of the extracellular domain of the epidermal growth factor receptor.

We studied the dimerization of the recombinant soluble extracellular domain of the epidermal growth factor receptor (sEGFR) in response to EGF-binding using multi-angle laser light scattering with size exclusion chromatography (SEC-MALLS). In the absence of EGF, sEGFR behaved as a monomer. However, upon EGF-binding, sEGFR formed a dimer with the stoichiometry of two EGF molecules bound to two sEGFR molecules [(EGF)2-(sEGFR)2]. We analyzed the chemical equilibrium of the dimer formation by SEC-MALLS using a dissociation constant of 0.25 microM for the binding of EGF to sEGFR. The calculated dissociation constant for EGF-induced sEGFR dimerization was found to be 2.4 +/- 0.9 microM. These experiments demonstrated that EGF induces receptor dimerization and that two EGF molecules are bound to an EGF-receptor dimer.

Binding Sites↗
PubMed1996

Structural characterization of a novel glycoinositolphospholipid from the parasitic nematode, Ascaris suum.

A novel glycosphingolipid containing inositol phosphate as an acidic group has been demonstrated in whole tissues of the porcine roundworm, Ascaris suum. The thin layer chromatographic pattern of the total acidic glycolipid revealed the presence of several components, of which a major component (named AGL) with positive reactions toward both orcinol-sulfuric acid (sugar) and molybdate (phosphate) spray reagents was isolated and purified by the use of successive column chromatography on DEAE-Sephadex and silicic acid (latrobeads). From structural studies including compositional sugar analysis, hydrogen fluoride degradation, methylation analysis, periodate oxidation, proton magnetic resonance spectroscopy and fast atom bombardment mass spectrometry, the structure of AGL was deduced to be Gal alpha 1-2Ins(1-->)-P-Cer. Aliphatic constituents were lignoceric acid and its 2-hydroxy homologue as the principal fatty acids, and octadecasphinganine and branched heptadecasphinganine as the major sphingoids.

Animals↗
PubMed1996

A novel fucosyltetrahexosylceramide in plerocercoids of the parasite Spirometra erinacei.

This glycosphingolipid was tentatively designated as GalSEGLx, in which the carbohydrate structure is characterized by an additional galactose molecule attached to the reducing-end galactose of SEGLx [Gal beta-4 (Fuc alpha-3) Glc beta-3 Gal beta Cer], which was previously determined by us [Kawakami, Y., Nakamura, K., Kojima, H., Suzuki, M., Inagaki, F., Suzuki, A., Sonoki, S., Uchida, A., Murata, Y. & Tamai, Y. (1993) J. Biochem. 114, 677-683], through a beta 1-6 linkage. The ceramide contained sphinganine and 4D-hydroxysphinganine in an about equimolar ratio, and a non-hydroxy fatty acid with carbon atoms ranging from 16 to 28, 26:0, 28:0 and 28:1 being major components. Based on the finding that a novel carbohydrate structure. Gal beta-4 Glc beta-3 Gal, was commonly found in glycosphingolipids from the parasite, S. erinacei, we here propose the terms, spirometo series, for this core structure series, and spirometosides, for glycosphingolipids having this carbohydrate structure.

Animals↗
PubMed1996

Tertiary structure of destrin and structural similarity between two actin-regulating protein families.

Destrin is an isoprotein of cofilin that regulates actin cytoskeleton in various eukaryotes. We determined the tertiary structure of destrin by triple-resonance multidimensional nuclear magnetic resonance. In spite of there being no significant amino acid sequence homology, we found that the folding of destrin was strikingly similar to that of repeated segments in the gelsolin family, which resulted in a new protein fold group. Sequential dissimilarity of the actin-binding helix of destrin to that of gelsolin explains the Ca2+-independent actin-binding of destrin. Possible mechanisms of phosphorylation-sensitive phosphoinositide-competitive actin binding, of pH-dependent filament severing, and of nuclear translocation with actin in response to stresses, are discussed on the basis of the tertiary structure.

Actin Depolymerizing Factors↗