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Mitsuhiro Kinoshita

Publications and source records attributed to Mitsuhiro Kinoshita.

At least 19 recordsLinked to original sources
PubMed2006

Analysis of total N-glycans in cell membrane fractions of cancer cells using a combination of serotonin affinity chromatography and normal phase chromatography.

Cell surface glycans and recognition molecules of these glycans play important roles in cellular recognition and trafficking, such as in the inflammation response by sialyl LewisX oligosaccharides. Malignant cells also utilize a similar mechanism during colonization and establishment of tumor tissues in the host. These considerations prompt us to develop a screening method for comprehensive analysis of N-glycans derived from membrane fractions of cancer cells. The method involves two step separations. Initially, N-glycans released from cell membrane fractions with N-glycoamidase F were labeled with 2-aminobenzoic acid and separated based on the number of sialic acid residues attached to the oligosaccharides using affinity chromatography on a serotonin-immobilized stationary phase. Each of the nonretarded fractions containing asialo- and high-mannose type oligosaccharides and mono-, di-, tri-, and tetra-sialooligosaccharide fractions which were desialylated with neuraminidase was analyzed by a combination of HPLC using an Amide-80 column as the stationary phase and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We analyzed total N-glycan pools of membrane fractions obtained from some cancer cells, and found that U937 cells (Histocytic lymphoma cells) expressed a large amount of oligosaccharides having polylactosamine residues and MKN45 cells (Gastric adenocarcinoma cells) contained hyper-fucosylated oligosaccharides which contained multiple fucose residues. The method described here will be a powerful technique for glycomics studies in cell surface glycoproteins, and will enable one to search marker oligosaccharides characteristically observed in various diseases such as cancer, inflammation, and congenital disorder.

Cell Membrane↗
PubMed2005

Capillary affinity electrophoresis using lectins for the analysis of milk oligosaccharide structure and its application to bovine colostrum oligosaccharides.

Animal colostrum and milk contain complex mixtures of oligosaccharides, which have species-specific profiles. Milk oligosaccharides have various types of structure related to the core structures of glycolipids and N- and O-glycans of glycoproteins and provide a good library to examine the binding of oligosaccharides to various lectins. Recently, we reported a capillary affinity electrophoresis (CAE) method for analyzing the interactions between lectins and complex mixtures of N-linked oligosaccharides prepared from serum glycoproteins. The present paper reports the interactions between 24 milk oligosaccharides and six lectins (PA-I, RCA(120), SBA, WGA, UEA-I, and AAL) analyzed using CAE. Based on the resulting data, we constructed a library that enables us to determine nonreducing terminal monosaccharides, such as Gal, GalNAc, GlcNAc, and Fuc, and to differentiate Gal- or Fuc-linked isomers, such as lacto-N-tetraose, lacto-N-neotetraose, and lacto-N-fucopentaose II and III. In addition, using the library, we show that a combination of the lectins can characterize the neutral oligosaccharides derived from bovine colostrum.

Animals↗
PubMed2005

Analysis of glycoprotein-derived oligosaccharides in glycoproteins detected on two-dimensional gel by capillary electrophoresis using on-line concentration method.

Capillary electrophoresis (CE) is an effective tool to analyze carbohydrate mixture derived from glycoproteins with high resolution. However, CE has a disadvantage that a few nanoliters of a sample solution are injected to a narrow capillary. Therefore, we have to prepare a sample solution of high concentration for CE analysis. In the present study, we applied head column field-amplified sample stacking method to the analysis of N-linked oligosaccharides derived from glycoprotein separated by two-dimensional gel electrophoresis. Model studies demonstrated that we achieved 60-360 times concentration effect on the analysis of carbohydrate chains labeled with 3-aminobenzoic acid (3-AA). The method was applied to the analysis of N-linked oligosaccharides from glycoproteins separated and detected on PAGE gel. Heterogeneity of alpha1-acid glycoprotein (AGP), i.e. glycoforms, was examined by 2D-PAGE and N-linked oligosaccharides were released by in-gel digestion with PNGase F. The released oligosaccharides were derivatized with 3-AA and analyzed by CE. The results showed that glycoforms having lower pI values contained a larger amount of tetra- and tri-antennary oligosaccharides. In contrast, glycoforms having higher pI values contained bi-antennary oligosaccharides abundantly. The result clearly indicated that the spot of a glycoprotein glycoform detected by Coomassie brilliant blue staining on 2D-PAGE gel is sufficient for quantitative profiling of oligosaccharides.

Carbohydrate Sequence↗
PubMed2005

Decreased mucin concentrations in tear fluids of contact lens wearers.

Characteristics of tear-film may be influenced by contact lens wear, because contact lenses present the habitual, direct rubbing action of the lids upon the covered ocular surface and may cause changes of tear-film. In the present paper, influence of contact lens on proteins in tear samples was studied using carbohydrates attached to the protein as a marker. We found that N-acetylneuraminic acid (Neu5Ac) was significantly decreased in tear samples of volunteers wearing contact lens (wearing, 86.1 +/- 57.7 nmol/ml; normal, 190.2 +/- 121.9 nmol/ml). Analysis by polyacrylamide gel electrophoresis demonstrated that the amounts of major proteins in tear fluids, such as lactoferrin and secretory immunoglobulin A were not changed upon wearing contact lenses. In contrast, cellulose acetate membrane electrophoresis revealed that mucin band in tear samples from contact lens wearers showed significant decrease as examined by lectin staining.

Chromatography, High Pressure Liquid↗
PubMed2005

Analysis of the interaction between hyaluronan and hyaluronan-binding proteins by capillary affinity electrophoresis: significance of hyaluronan molecular size on binding reaction.

We developed a method for the analysis of the interaction between hyaluronan (HA) oligosaccharides and hyaluronan-binding proteins (HABPs) using capillary affinity electrophoresis (CAE). The method is based on high-resolution separation of fluorescent-labeled HA molecules in the presence of hyaluronan-binding proteins at different concentrations by capillary electrophoresis (CE) with laser-induced fluorescent detection. Hyaluronan-binding protein from bovine nasal cartilage interacts strongly with HA decasaccharide or larger oligosaccharides. Effect of the molecular size of HA oligomers clearly showed that longer carbohydrate chains than decasaccharide were required for recognition by HA binding protein. Interestingly, the interaction did not cause retardation of HA oligomers as observed in many binding reactions such as the interaction between pharmaceuticals and serum albumin, but showed disappearance of the oligomer peak. Although we cannot explain the accurate mechanism on the interaction, disappearance is probably due to low equilibrium rate between free and conjugate states. The present technique will be useful to compare the relative binding affinity, and to understand the mechanism on the interaction between hyaluronan and hyaluronan-binding proteins.

Animals↗
PubMed2004

Fast analysis of glycosaminoglycans by microchip electrophoresis with in situ fluorescent detection using ethidium bromide.

We analyzed some glycosaminoglycans and natural and artificial acidic polysaccharides using microchip electrophoresis in the buffer containing ethidium bromide, and found that they were successfully separated and detected within 150 s with comparable sensitivity with that of conventional electrophoresis using cellulose acetate membrane. We applied the technique to the analysis of glycosaminoglycans in pharmaceutical preparations and also in cultured cancer cells. Rapidness and easy operation of the proposed technique are quite useful for routine analysis of glycosaminoglycans.

Animals↗
PubMed2004

Profiling analysis of oligosaccharides in antibody pharmaceuticals by capillary electrophoresis.

Carbohydrate chains in glycoprotein pharmaceuticals have important roles for the expression of their biological activities. Therefore, development of an assessment method for the carbohydrate chains is an important parameter for quality control of glycoprotein pharmaceuticals such as newly developed therapeutic antibodies. In this report, we applied capillary electrophoresis with laser-induced fluorescence detection to the analysis of carbohydrate chains after releasing with glycoamidase followed by derivatization with 3-aminobenzoic acid. We found that four major oligosaccharides present in antibody pharmaceuticals were successfully separated with good resolution. The present method showed good precision in both migration times and relative peak areas, and gave comparable accuracy with that using a derivatization method with 8-aminopyrene-1,3,6-trisulfonate.

Aminobenzoates↗
PubMed2004

Electrophoretic analysis of di- and oligosaccharides derived from glycosaminoglycans on microchip format.

Microchip electrophoresis is a powerful tool for fast analysis of nucleic acids and has expanded its applicability to the analysis of various biological materials including proteins and carbohydrates. Glycosaminoglycans have intrinsic negative charges, and are good targets for electrophoretic analysis. In the present paper, we developed a method to analyze oligosaccharides and unsaturated disaccharides derived from some glycosaminoglycans after digestion with specific enzymes followed by derivatization with 2-aminoacrydone (AMAC) by reductive amination. The method described here allowed rapid analysis of oligosaccharides derived from glycosaminoglycans within 150 s with high sensitivity. We show an application of the present technique to the glycosaminoglycan analysis in cultured HeLa cells.

Aminoacridines↗
PubMed2004

Screening method of carbohydrate-binding proteins in biological sources by capillary affinity electrophoresis and its application to determination of Tulipa gesneriana agglutinin in tulip bulbs.

We developed capillary affinity electrophoresis (CAE) to analyze the molecular interaction between carbohydrate chains and proteins in solution state. A mixture of oligosaccharides derived from a glycoprotein was labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS), and used as glycan library without isolation. Interaction of a carbohydrate-binding protein with each oligosaccharide in the mixture could be simultaneously observed, and relative affinities of oligosaccharides toward the protein were accurately determined. In this study, we applied CAE to detect the presence of lectins in some plants (Japanese elderberry bark and tulip bulb). In the crude extract of the elderberry bark, binding activity toward sialo-carbohydrate chains could be easily detected. We also examined the presence of lectins in the crude extract of tulip bulbs and determined the detailed carbohydrate-binding specificity of Tulipa gesneriana agglutinin (TGA), one of the lectins from tulip bulbs. Kinetic studies demonstrated that TGA showed novel carbohydrate-binding specificity and preferentially recognized triantennary oligosaccharides with Gal residues at nonreducing termini and a Fuc residue linked through alpha(1-6) linkage at chitobiose portion of the reducing termini but not tetraantennary carbohydrates. The results described here indicate that CAE will be a valuable method for both screening of lectins in natural sources and determination of their detailed carbohydrate-binding specificities.

Carbohydrate Sequence↗
PubMed2004

Capturing of acidic macromolecules from biological samples using a temperature-responsive polymer modified with poly-l-lysine.

We synthesized a temperature-responsive polymer, N-(isopropylacrylamide)-methacrylic acid copolymer, to which poly-l-lysine was introduced. The synthesized polymer as well as the parent polymer showed reversible soluble-insoluble changes in response to temperature changes across the lower critical solution temperature at 32 degree C in an aqueous solution. We found that the polymer efficiently captured acidic bio-macromolecules such as RNA, glycosaminoglycans and mucin-type glycoproteins in biological samples, and the captured molecules were recovered using aqueous NaCl solutions at high concentration. The target acidic molecules thus obtained will be employed for further studies such as structural analysis after brief desalting procedure. The proposed method does not require any chromatographic separations, but only needs a small volume of an aqueous salt solution for releasing captured molecules. Overall procedures are quite easy and simple, and are completed at least within 1 h. We show a few examples for capturing RNA and glycosaminoglycans from cultured cells using the polymer.

Acrylamides↗
PubMed2004

A protective role of protease-activated receptor 1 in rat gastric mucosa.

BACKGROUND AND AIMS: On activation, protease-activated receptor (PAR)-2 modulates multiple gastric functions and exerts mucosal protection via activation of sensory neurons. The role of PAR-1, a thrombin receptor, in the stomach remains unknown. We thus examined if the PAR-1 agonist could protect against gastric mucosal injury in rats. METHODS: Gastric mucosal injury was created by oral administration of ethanol/HCl or absolute ethanol in conscious rats. Gastric mucosal blood flow and acid secretion were determined in anesthetized rats. Immunohistochemical analyses of PAR-1 and cyclooxygenase (COX)-1 were also performed in rat and human stomach. RESULTS: The PAR-1 agonist TFLLR-NH(2), administered intravenously in combination with amastatin, protected against the gastric mucosal injury induced by ethanol/HCl or absolute ethanol. The protective effect of TFLLR-NH(2) was abolished by indomethacin or a COX-1 inhibitor but not by ablation of sensory neurons with capsaicin. TFLLR-NH(2) produced an NO-independent increase in gastric mucosal blood flow that was partially inhibited by blockade of the endothelium-derived hyperpolarizing factor pathway. This inhibitory effect was promoted by indomethacin. TFLLR-NH(2) suppressed carbachol-evoked acid secretion in an indomethacin-reversible manner. Immunoreactive PAR-1 and COX-1 were expressed abundantly in rat gastric muscularis mucosae and smooth muscle, and the former protein was also detectable in blood vessels. Similar staining was observed in human gastric muscularis mucosae. CONCLUSIONS: The PAR-1 agonist, given systemically, protects against gastric mucosal injury via COX-1-dependent formation of prostanoids, modulating multiple gastric functions. Our data identify a novel protective role for PAR-1 in gastric mucosa, and the underlying mechanism is entirely different from that for PAR-2.

Animals↗
PubMed2003

Hyaluronic acid: separation and biological implications.

Hyaluronic acid (hyaluronan) is a ubiquitous extracellular matrix component, and present at high concentrations in skin, joints and cornea. In the skin, it is synthesized primarily by dermal fibroblasts and by epidermal keratinocytes. Hyaluronic acid usually exists as a high molecular mass (600,000-1,000,000) and non-sulfated glycosaminoglycan composed of a disaccharide unit of [bond]3GlcNAc beta 1[bond]4GlcA beta 1[bond]. Hyaluronic acid has been widely used not only for osteoarthritis and ophthalmology but also for cosmetics for skin care. To examine the biological activities of hyaluronic acid, we have to accurately determine the quantity and molecular masses in biological samples. We review recent development in the analysis of hyaluronic acid having various molecular sizes using electrophoretic and chromatographic techniques. Recently, interactions between hyaluronic acid oligomers and hyaluronic acid-binding proteins have attracted the interest for understanding the biological functions. We show some interesting reports on biological interactions of hyaluronic acid and its oligomers with some proteins.

Chromatography, Liquid↗
PubMed2003

A new fungal lectin recognizing alpha(1-6)-linked fucose in the N-glycan.

In this report, we describe a new lectin from the fungus Rhizopus stolonifer that agglutinates rabbit red blood cells. Agglutinating activity was detected in the extract of mycelium-forming spores cultured on agar plates but not in the mycelium-forming no spores from liquid medium. This lectin, which we designated R. stolonifer lectin (RSL), was isolated by affinity chromatography with porcine stomach mucin-Sepharose. SDS-polyacrylamide gel electrophoresis and mass spectral analysis showed that RSL is approximately 4.5 kDa, whereas gel filtration indicated a mass of 28 kDa. This indicates that the lectin is a hexamer of noncovalently associated RSL monomers. RSL activity was very stable, since it was insensitive to heat treatment at 70 degrees C for 10 min. Analysis of RSL binding specificity by both microtiter plate and precipitation assays showed that N-glycans with l-fucose linked to the reducing terminal GlcNAc residues are the most potent inhibitors of RSL binding, whereas N-glycans without alpha(1-6)-linked fucose residues are approximately 100-fold weaker inhibitors of binding. Oligosaccharides with alpha(1-2, -3, and -4) linkages showed no inhibition of binding in these assays. In a mirror resonance biosensor assay, high affinity binding was observed between RSL and the glycopeptide of bovine gamma-globulin, which has N-glycans with alpha(1-6)-linked fucose residues. However, RSL showed only a weak interaction with the glycopeptide of quail ovomucoid, which lacks fucose residues. Finally, capillary affinity electrophoresis studies indicated that RSL binds strongly to N-glycans with alpha(1-6)-linked fucose residues. Together, these results show that RSL recognizes the core structure of N-glycans with alpha(1-6)-linked l-fucose residues. This specificity could make RSL a valuable tool for glycobiological studies.

Amino Acid Sequence↗
PubMed2003

A method for detecting O-glycanase in biological samples using a combination of MALDI-TOF mass spectrometry and time-resolved fluorimetry.

O-Glycans (mucin type oligosaccharides) are ubiquitously found in various glycoproteins such as mucin-type glycoproteins on the surface of various digestive organs. In the present paper, we propose a method for detecting O-glycanase which catalyzes the hydrolysis of the O-glycan linkage between oligosaccharides and serine/threonine residues of mucin-type glycoprotein. As the substrate for O-glycanases, we chose glycopeptides containing O-glycans derived from bovine fetuin. The present method is divided into two parts. At the initial stage, the presence of O-glycanase was confirmed by observing characteristic ions due to O-glycans and peptides released from the glycopeptide by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In the second step, europium labeled O-glycosylated peptide permits more detailed analysis such as enzyme kinetics. We demonstrated the usefulness of the present method using O-glycanase (Streptococcus pneumoniae) as model enzyme. The present approach can easily confirm the presence of O-glycanase by detecting both deglycosylated peptides and O-glycans, even if contaminating peptides or glycosidases are present in crude biological samples.

Amino Acid Sequence↗
PubMed2002

Collection of alpha1-acid glycoprotein molecular species by capillary electrophoresis and the analysis of their molecular masses and carbohydrate chains. Basic studies on the analysis of glycoprotein glycoforms.

A highly heterogeneous glycoprotein, alpha1-acid glycoprotein, was resolved into their glycoforms by capillary electrophoresis using a surface-modified capillary in 20 mM acetate buffer (pH 4.2) containing 0.5% (w/v) hydroxypropylmethylcellulose. We collected the fractions containing each glycoform as nearly pure state by capillary electrophoresis, and examined the molecular masses of these glycoforms by matrix assisted laser desorption time-of-flight mass spectrometry. We also analyzed carbohydrate chains after releasing them with N-glycosidase F followed by fluorescent labeling with 8-aminopyrene-1,3,6-trisulfonate. We found that the separation of glycoforms was mostly due to the presence of multiantennary carbohydrate chains. We propose that the present technique is useful for the analysis of post translational modification of proteins with carbohydrate chains.

Carbohydrate Sequence↗
PubMed2002

Determination of molecular mass of acidic polysaccharides by capillary electrophoresis.

We developed a method for the determination of molecular mass of acidic polysaccharides based on their high-resolution separation by capillary electrophoresis. Polymers of N-acetylneuraminic acid (NeuAc) and polysulfated hyaluronic acid were separated into their molecular species up to 100 mono- and 20 disaccharide units, respectively. The relationship between the molecular mass of NeuAc-polymer and their electrophoretic mobilities showed good linearity, and was applied to the determination of molecular masses of larger NeuAc species unresolved by capillary electrophoresis under the same conditions. In the first step, the standard curve for the determination of molecular mass was constructed from the relationship between electrophoretic mobility and molecular mass. Subsequently, the mobility was extrapolated to the standard curve, and the molecular mass was calculated. Five different preparations of NeuAc polymers having different molecular masses showed smaller values than those determined by conventional chromatographic techniques. Further, molecular mass determined by the present method correlated with number-average molecular mass. The methodology presented here was applied to the determination of molecular mass of polysulfated hyaluronic acid. The data indicated that native hyaluronic acid was extensively degraded during sulfonation reaction.

Acids↗
PubMed2002

Analysis of glycoproteins and the oligosaccharides thereof by high-performance capillary electrophoresis-significance in regulatory studies on biopharmaceutical products.

This review describes the recent development in the analysis of glycoproteins using capillary electrophoresis with various separation techniques, and focuses especially on the analysis of recombinant glycoprotein pharmaceuticals. We include the analysis of glycoprotein multiforms (ie glycoform) as well as glycan analysis. The relationship between glycoprotein multiforms and oligosaccharide distributions in a glycoprotein sample is also discussed. Further, recent development in capillary electrophoresis-mass spectrometry is described.

Carbohydrate Sequence↗