[Epidermal growth factor receptor].
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Amino Acid Sequence↗
Biomedical subjects
F Inagaki
Publications and source records attributed to F Inagaki.
Novel gangliosides containing the sialyl-Le(a) structure from a human rectal adenocarcinoma.
Oligosaccharides were released from the gangliosides of a human rectal adenocarcinoma with endoglycoceramidase (Rhodococcus sp. G-74-2) and then purified by affinity chromatography on a column of an immobilized monoclonal antibody, MSW 113. Structural studies, involving 600-MHz 1H NMR spectrometry, indicated the structures of these compounds to be as follows. [formula: see text] Three of these oligosaccharides, 2, 3, and 4, are novel as to ganglioside sugar chains and contain both lacto series types 1 and 2 chains. Oligosaccharides 3 and 4 are unique in that they contain the sialyl-Le(a)-X structure in linear and branched structures. Gangliosides with the oligosaccharide structures presented above might be new potential tumor markers.
Adenocarcinoma↗
A bacterium lipopolysaccharide that elicits Guillain-Barré syndrome has a GM1 ganglioside-like structure.
There is a strong association between Guillain-Barré syndrome (GBS) and Penner's serotype 19 (PEN 19) of Campylobacter jejuni. Sera from patients with GBS after C. jejuni infection have autoantibodies to GM1 ganglioside in the acute phase of the illness. Our previous work has suggested that GBS results from an immune response to cross-reactive antigen between lipopolysaccharide (LPS) of the Gram-negative bacterium and membrane components of peripheral nerves. To clarify the pathogenesis of GBS, we have investigated whether GM1-oligosaccharide structure is present in the LPS of C. jejuni (PEN 19) that was isolated from a GBS patient. After extraction of the LPS, the LPS showing the binding activity of cholera toxin, that specifically recognizes the GM1-oligosaccharide was purified by a silica bead column chromatography. Gas-liquid chromatography-mass spectrometric analysis has shown that the purified LPS contained Gal, GalNAc, and NeuAc, which are sugar components of GM1 ganglioside. 1H NMR methods [Carr-Purcell-Meiboom-Gill (CPMG), total correlation spectroscopy (TOCSY), and nuclear Overhauser effect spectroscopy (NOESY)] have revealed that the oligosaccharide structure [Gal beta 1-3 GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta] protrude from the LPS core. This terminal structure [Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta] is identical to the terminal tetrasaccharide of the GM1 ganglioside. This is the first study to demonstrate the existence of molecular mimicry between nerve tissue and the infectious agent that elicits GBS.
Adult↗
Solution structure of the SH3 domain of phospholipase C-gamma.
SH3 (Src homology 3) domains are found in many signaling proteins and appear to function as binding modules for cytoplasmic target proteins. The solution structure of the SH3 domain of human phospholipase C-gamma (PLC-gamma) was determined by two-dimensional 1H NMR analysis. This SH3 domain is composed of eight antiparallel beta strands consisting of two successive "Greek key" motifs, which form a barrel-like structure. The conserved aliphatic and aromatic residues form a hydrophobic pocket on the molecular surface, and the conserved carboxylic residues are localized to the periphery. The hydrophobic pocket may serve as a binding site for target proteins. Analysis of the slowly exchanging amide protons by NMR measurements indicates that despite containing a high content of beta structure, the SH3 domain of PLC-gamma is flexible.
Amino Acid Sequence↗
A 40-kDa epidermal growth factor/transforming growth factor alpha-binding domain produced by limited proteolysis of the extracellular domain of the epidermal growth factor receptor.
Elucidation of the three-dimensional structure of the complex of the epidermal growth factor (EGF) and its receptor is essential for understanding the molecular mechanisms of the EGF-receptor interaction and EGF-induced receptor-receptor interaction. NMR is useful to investigate interactions in solution between macromolecules at atomic resolution, but has a limitation in molecular masses of target proteins: less than 300 residues. We have prepared a fragment with apparent molecular mass of 40 kDa in SDS gels from the soluble extracellular domain of the EGF receptor (sEGFR, 619 residues) by sequential limited proteolysis with proteinase K and bromelain. This fragment is a monomeric structural domain consisting of 202 amino acid residues (Cys302-Arg503) and 18-kDa sugar chains, and binds EGF and transforming growth factor-alpha (TGF alpha). This 40-kDa domain has a dissociation constant of about 1 microM for human TGF alpha, which is similar to that of the parental sEGFR. sEGFR oligomerizes in response to EGF and TGF alpha, while the 40-kDa domain does not, suggesting that the sequences other than this domain is required for receptor oligomerization. The 40-kDa ligand-binding domain described in this report is suitable for analysis by various physico-chemical approaches such as NMR.
Amino Acid Sequence↗
Secondary structural features of modules M2 and M3 of barnase in solution by NMR experiment and distance geometry calculation.
Proteins consist of structural units such as globular domains, secondary structures, and modules. Modules were originally defined by partitioning a globular domain into compact regions, each of which is a contiguous polypeptide segment having a compact conformation. Since modules show close correlations with the intron positions of genes, they are regarded as primordial polypeptide pieces encoded by exons and shuffled, leading to yield new combination of them in early biological evolution. Do modules maintain their native conformations in solution when they are excised at their boundaries? In order to find answers to this question, we have synthesized modules of barnase, one of the bacterial RNases, and studied the solution structures of modules M2 (amino acid residues 24-52) and M3 (52-73) by 2D NMR studies. Some local secondary structures, alpha-helix, and beta-turns in M2 and beta-turns in M3, were observed in the modules at the similar positions to those in the intact barnase but the overall state seems to be in a mixture of random and native conformations. The present result shows that the excised modules have propensity to form similar secondary structures to those of the intact barnase.
Amino Acid Sequence↗
Novel oligosaccharides with the sialyl-Le(a) structure in human milk.
Two novel oligosaccharides with the sialyl-Le(a) structure were isolated from human milk using a monoclonal antibody, MSW 113. These oligosaccharides were purified by affinity chromatography on a column of the immobilized monoclonal antibody and by high-performance liquid chromatography, and structurally characterized by a combination of 600-MHz 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The structural studies indicated the structures of these oligosaccharides to be: [formula: see text]
Antibodies, Monoclonal↗
A novel fucosylated glycosphingolipid with a Gal beta 1-4Glc beta 1-3Gal sequence in plerocercoids of the parasite, Spirometra erinacei.
A novel glycosphingolipid (SEGLx) has been isolated from the plerocercoids of a tapeworm, Spirometra erinacei. From the results of compositional analysis, methylation analysis, exoglycosidase hydrolysis, acid hydrolysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance (NMR) analysis, its structure was concluded to be [formula: see text] This is the first report of a glycosphingolipid with a novel carbohydrate structure which is characterized by i) the occurrence of a penultimate glucose molecule attached to the reducing end galactose through a beta 1-3 linkage and ii) the presence of a fucose attached to a glucose through an alpha 1-3 linkage. The ceramide contained sphinganine or 4-D-hydroxy-sphinganine, and either a nonhydroxy fatty acid with 16, 18, 26, or 28 carbon atoms, or hydroxystearic acid. Proton NMR analysis revealed that the chemical species of both the long chain base and fatty acid moieties affect the chemical shifts of the anomeric proton resonances of not only the reducing terminal galactose but also the penultimate glucose.
Animals↗
Structure-activity relationships of mu-conotoxin GIIIA: structure determination of active and inactive sodium channel blocker peptides by NMR and simulated annealing calculations.
A synthetic replacement study of the amino acid residues of mu-conotoxin GIIIA, a peptide blocker for muscle sodium channels, has recently shown that the conformation formed by three disulfide bridges and the molecular basicity, especially the one around the Arg13 residue, are important for blocking activity. In the present study, we determined the three-dimensional structure of an inactive analog, [Ala13]mu-conotoxin GIIIA, and refined that of the native toxin by NMR spectroscopy combined with simulated annealing calculations. The atomic root-mean-square difference of the mutant from the native conotoxin was 0.62 A for the backbone atoms (N, C alpha, C') of all residues except for the two terminal residues. The observation that the replacement of Arg13 by Ala13 does not significantly change the molecular conformation suggests that the loss of activity is not due to the conformational change but to the direct interaction of the essential Arg13 residue with the sodium channel molecules. In the determined structure, important residues for the activity, Arg13, Lys16, Hyp(hydroxyproline)17, and Arg19, are clustered on one side of the molecule, an observation which suggests that this face of the molecule associates with the receptor site of sodium channels. The hydroxyl group of Hyp17 is suggested to interact with the channel site with which the essential hydroxyl groups of tetrodotoxin and saxitoxin interact.
Alanine↗
Three-dimensional nuclear magnetic resonance structures of mouse epidermal growth factor in acidic and physiological pH solutions.
The three-dimensional structures of epidermal growth factors (EGF) previously reported were all in acidic solutions (pH 2.0-3.2), at which pHs EGF cannot bind to the receptor. Here we studied the structure of mouse EGF at pH 6.8, where EGF is physiologically active, and compared it with the structure at pH 2.0 by CD and NMR. From pH dependence of CD spectra and a comparison between the chemical shifts of the proton resonances at pH 6.8 and 2.0, the conformations at two pHs were found to be nearly identical except for the C-terminal tail region. The three-dimensional structures at pH 6.8 and 2.0 were determined independently by a combination of two-dimensional 1H NMR and stimulated annealing calculations using the program XPLOR. The calculations were based on 261 distance constraints at pH 6.8 and 355 distance and 24 torsion angle constraints at pH 2.0. The conformational difference of the C-terminal domain (residues 33-50) was detected between the two structures, which were supported by CD and the chemical shift comparison. The positions of the side chains of Leu47, Arg48, Trp49, and Trp50 are changed probably by the effect of the deprotonation of Asp46. Considering the fact that Leu47 is essential in EGF binding to the receptor, this conformational difference may be important in receptor recognition.
Amino Acid Sequence↗
Polar glycosphingolipids in annelida. A novel series of glycosphingolipids containing choline phosphate from the earthworm, Pheretima hilgendorf.
A novel series of glycosphingolipids containing choline phosphate has been demonstrated in whole tissues of the earthworm, Pheretima hilgendorfi. The thin layer chromatographic pattern of the total polar glycolipids revealed the presence of more than three components with positive reactions toward orcinol-sulfuric acid (sugar), molybdate (phosphate), and Dragendorff's (choline) spray reagents. Two of these polar glycolipids (PGL1 and PGL2) were purified by the use of successive column chromatography on QAE-Sephadex A-25 and silicic acid (Iatrobeads) and detected during elution by the presence of galactose-bound choline phosphate. The structural elucidation of the oligosaccharide moieties was performed by compositional sugar analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, and methylation analysis. Thus, the structures of PGL1 and PGL2 were deduced to be as follows: cholinephosphoryl-->6Gal beta 1-1Cer and cholinephosphoryl-->6Gal beta 1-6Gal beta 1-1Cer. Although the oligosaccharide structures of both PGL1 and PGL2 have previously been found in other organisms, the presence of a choline phosphate group as an oligosaccharide substituent is the first finding in nature. The main molecular species of the ceramide moieties were composed of beheninyl- and lignocerinyloctadecasphingenines and their nonadecasphingenine homologues.
Animals↗
A novel disialoganglioside in rat spleen lymphocytes.
A novel ganglioside has been identified as the predominant disialoganglioside of the lymphocytes prepared from rat spleen. The ganglioside was isolated from rat spleen and characterized by compositional analysis, methylation analysis, sialidase hydrolysis, proton NMR spectroscopy, and negative ion fast atom bombardment mass spectrometry. The structure was determined as follows. [formula: see text] This ganglioside is a unique derivative of N-acetyllactosaminyl-GM1. The three monosialogangliosides containing N-acetyllactosaminyl-GM1 structure, which had been originally isolated from rat spleen (Nohara, K., Suzuki, M., Inagaki, F., Ito, H., and Kaya, K. (1990) J. Biol. Chem. 265, 14335-14339), were also found in the lymphocytes and were hardly detected in the spleen remnant tissue depleted of single cells. On the other hand, GD1c(NeuGc,NeuGc) (IV3(NeuGc alpha 2-8NeuGc)-Gg4Cer), the overwhelmingly predominant ganglioside of rat thymocytes (Nohara, K., Suzuki, M., Inagaki, F., and Kaya, K. (1991) J. Biochem. (Tokyo) 110, 274-278), was demonstrated to be only a minor component of the gangliosides of the spleen lymphocytes. These results suggested that GD1c is characteristic for the immature T lineage lymphoid cells and the gangliosides having lactosaminyl-GM1 structure are specific for other populations of the lymphocytes in rat.
Animals↗
Stable isotope aided nuclear magnetic resonance study to investigate the receptor-binding site of human interleukin 1 beta.
Resonance assignments for interleukin 1 beta at neutral pH were made by using three-dimensional NMR in combination with specific labeling and double-labeling methods with stable isotopes. On the basis of the present assignments, 15N single-quantum coherence spectra of N-terminal truncated and fusion mutants were compared with that of the wild-type. Although these mutants have reduced biological activity, they showed 15N-SQC spectra similar to that of the wild-type. However, small but significant chemical shift changes were observed for amino acid residues within a loop 86-99, in spite of the modification at the N-terminus, supporting the idea that this loop forms a biologically active part of interleukin 1 beta. Receptor-binding activity was studied for mutants (Asp-93)-, (Leu-93)- and des-(Arg-98)interleukin 1 beta's. The results show significant loss of the receptor-binding activity. The N-terminus, the C-terminus, and the loop 86-99 form a part of the open end of a beta-barrel [Finzel, B. C., Clancy, L. L., Holland, D. R., Muchmore, S. W., Watenpaugh, K. D., & Einspahr, H. M. (1989) J. Mol. Biol. 209, 779-791; Clore, G. M., Wingfield, P. T., & Gronenborn, A. M. (1991) Biochemistry 30, 2315-2323], which forms the receptor-binding site of IL-1 beta.
Amides↗
A novel 15N-labeling method to selectively observe 15NH2 resonances of proteins in 1H-detected heteronuclear correlation spectroscopy.
An experimental method to selectively label side-chain NH2 groups of glutamine and asparagine in proteins with 15N is proposed. This selective labeling method enables to observe only 15NH2 resonances and thus, to discriminate between 15NH and 15NH2 resonances in a 1H-detected heteronuclear correlation spectrum. This method gives results with approximately two times higher sensitivity than those obtained by elaborate pulse sequences such as DEPT-HSQC and will be useful for studying the molecular interaction involving the side chains of Asn and Gln residues.
Fibroblast Growth Factor 1↗
Structure of epidermal growth factor bound to perdeuterated dodecylphosphocholine micelles determined by two-dimensional NMR and simulated annealing calculations.
The interaction of mouse epidermal growth factor (mEGF) with micelles of a phospholipid analogue, perdeuterated dodecylphosphocholine (DPC), was investigated by two-dimensional 1H NMR. Sequence-specific resonance assignments of the micelle-bound mEGF have been made, and the chemical shifts were compared with those in the absence of DPC. DPC induced large chemical shift changes of the resonances from the residues in the C-terminal tail (residues 46-53) but little perturbation on the residues in the main core (residues 1-45). Starting from the three-dimensional structure in the absence of DPC, micelle-bound structures were calculated using the program XPLOR with interproton distance data obtained from NOESY spectra recorded in the presence of DPC. The C-terminal tail of mEGF was found to change conformation to form an amphiphilic structure when bound to the micelles. It is possible that induced fit in the C-terminal tail of mEGF occurs upon binding to a putative hydrophobic pocket of the EGF receptor.
Amino Acid Sequence↗
A trisialoganglioside containing a sialyl alpha 2-6 N-acetylgalactosamine residue is a cholinergic-specific antigen, Chol-1 alpha.
Cholinergic-specific antigens termed the Chol-1 family have been suggested to be of a ganglioside nature by Richardson et al. (J. Neurochem. 38, 1605-1614, 1982). Two molecular species of polysialogangliosides among bovine brain gangliosides were found to react with anti-Chol-1 alpha antiserum. One of them, Chol-1 alpha-a, was isolated and characterized as a trisialoganglioside containing the gangliotetraose backbone in which 1 mol of sialic acid was attached to each of the reducing end galactose, N-acetylgalactosamine and internal galactose residues, respectively. The chemical structure of Chol-1 alpha-a was determined for the first time, being as follows: IV3NeuAc III6NeuAc II3NeuAc-GgOse4 Cer.
Antigens, Surface↗
Novel structure of the N-acetylgalactosamine containing N-glycosidic carbohydrate chain of batroxobin, a thrombin-like snake venom enzyme.
The structure of the Asn-linked carbohydrate chain of batroxobin, a thrombin-like enzyme from Bothrops atrox moojeni snake venom, has been determined. The sugar chain was isolated from batroxobin by hydrazinolysis followed by pyridylamination (PA). The PA-oligosaccharide chain was purified by HPLC on an anion exchange or reverse phase columns, and its structure was examined by sequential exoglycosidase digestion, 600 MHZ 1H NMR spectroscopy and methylation analysis. The results indicate that the oligosaccharide chain has the following structure involving a novel linkage, NeuAc alpha 2----3GalNAc.
Acetylgalactosamine↗
A novel difucosylated neutral glycosphingolipid from the eggs of the sea urchin, Hemicentrotus pulcherrimus: I. Purification and structural determination of the glycolipid.
A novel fucose-containing neutral glycosphingolipid (GL-5) was purified from the eggs of the sea urchin, Hemicentrotus pulcherrimus. The chemical structure was determined to be Fuc alpha 1-3GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-4Glc beta 1-1Cer by methylation analysis, partial acid hydrolysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The unique characteristics of GL-5 are that: the reducing terminal disaccharide portion is not Gal beta 1-4Glc but GlcNAc beta 1-4Glc; it includes a GalNAc beta 1-4GlcNAc sequence and a Fuc-GalNAc linkage; the defucosylated core is a novel trisaccharide chain; and the sugar structure is one of the smallest ever characterized for a difucosylated glycolipid. The major fatty acids were 22:1 and 22h:1, and about 30% of the total acids was 2-hydroxylated. All the long-chain bases were phytosphingosines, of which about 90% was n-t18:0. The similarity of the ceramide moiety to that of glucosylceramide from the same eggs [Kubo, H. et al. (1992) J. Biochem. 111, 726-731] suggests a close biosynthetic relationship between GL-5 and the glucosylceramide.
Animals↗