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Miyako Ohta

Publications and source records attributed to Miyako Ohta.

9 recordsLinked to original sources
PubMed2004

Analysis of site-specific glycosylation in recombinant human follistatin expressed in Chinese hamster ovary cells.

Follistatin (FS), a glycoprotein, plays an important role in cell growth and differentiation through the neutralization of the biological activities of activins. In this study, we analyzed the glycosylation of recombinant human FS (rhFS) produced in Chinese hamster ovary cells. The results of SDS-PAGE and MALDI-TOF MS revealed the presence of both non-glycosylated and glycosylated forms. FS contains two potential N-glycosylation sites, Asn95 and Asn259. Using mass spectrometric peptide/glycopeptide mapping and precursor-ion scanning, we found that both N-glycosylation sites were partially glycosylated. Monosaccharide composition analyses suggested the linkages of fucosylated bi- and triantennary complex-type oligosaccharides on rhFS. This finding was supported by mass spectrometric oligosaccharide profiling, in which the m/z values and elution times of some of the oligosaccharides from rhFS were in good agreement with those of standard oligosaccharides. Site-specific glycosylation was deduced on the basis of the mass spectra of the glycopeptides. It was suggested that biantennary oligosaccharides are major oligosaccharides located at both Asn95 and Asn259, whereas the triantennary structures are present mainly at Asn95.

Amino Acid Sequence↗
PubMed2004

Enhancement of hepatocyte growth factor-induced cell scattering in N-acetylglucosaminyltransferase III-transfected HepG2 cells.

N-Acetylglucosaminyltransferase III (GnT-III), which catalyzes the synthesis of a bisecting GlcNAc residue of N-glycans, is thought to be involved in the function of glycoproteins such as growth factor receptors. We investigated the effects of the overexpression of GnT-III on the hepatocyte growth factor (HGF) receptor c-Met, a glycoprotein, in human hepatocarcinoma HepG2 cells. GnT-III activity was elevated about 250-fold in HepG2 cells stably transfected with the GnT-III gene, whereas no significant change in GnT-III activity was observed in mock transfectants. Cell scattering assay revealed that HGF-induced cell scattering was enhanced depending on the GnT-III activities in the GnT-III transfectants. Western blot analysis and E-PHA lectin blot analysis showed that the level of c-Met protein was the same in both transfectants; however, the bisecting GlcNAc residue on c-Met was detected only in the GnT-III transfectants. Although the peak level of c-Met phosphorylation was not different in both transfectants, the level of tyrosine phosphorylation of c-Met decreased more rapidly in the GnT-III transfectants than in the mock transfectants. Furthermore, HGF-induced extracellular-regulated kinase (ERK) phosphorylation was slightly higher in the GnT-III transfectants than in the mock transfectants. These results show that overexpression of GnT-III in HepG2 cells enhances HGF-induced cell scattering, which may result from, at least in part, enhancement of HGF-induced ERK phosphorylation.

Cell Line, Tumor↗
PubMed2003

Microanalysis of N-linked oligosaccharides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry.

We have studied rapid and simple sugar mapping using liquid chromatography/electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column. The oligosaccharide mixture was separated on the basis of the sequence, branching structure, and linkage, and each oligosaccharide was characterized based on its molecular mass. In this study we demonstrated the usefulness of capillary LC/MS (CapLC/MS) and capillary liquid chromatography/tandem mass spectrometry (CapLC/MS/MS) as sensitive means for accomplishing the structural analysis of oligosaccharides in a low-abundance glycoprotein. The carbohydrate heterogeneity and molecular mass information of each oligosaccharide can be readily obtained from CapLC/MS of a small amount of glycoprotein. CapLC/MS/MS provided b-ion series, which is informative with regard to monosaccharide sequence. Exoglycosidase digestion followed by CapLC/MS elucidated a carbohydrate residue linkage. Using this method, we characterized N-linked oligosaccharides in hepatocyte growth factor produced in mouse myeloma NS0 cells as the complex-type bi-, tri-, and tetraantennary terminated with N-glycolylneuraminic acids and alpha-linked galactose residues. Sugar mapping with CapLC/MS and CapLC/MS/MS is useful for monitoring glycosylation patterns and for structural analysis of carbohydrates in a low-abundance glycoprotein and thus will become a powerful tool in biological, pharmaceutical, and clinical studies.

Animals↗
PubMed2002

Structural analysis of a glycoprotein by liquid chromatography-mass spectrometry and liquid chromatography with tandem mass spectrometry. Application to recombinant human thrombomodulin.

Using recombinant human thrombomodulin (rhTM) expressed in Chinese hamster ovary (CHO) cells, we studied the structural analysis of a glycoprotein by liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS-MS). First, we analyzed the structure of both the O- and N-linked glycans in rhTM by oligosaccharide mapping using LC-MS equipped with a graphitized carbon column (GCC-LC-MS). Major O- and N-linked glycans were determined to be core 1 structure and fucosyl biantennary containing NeuAc(0-2) respectively. Next, the post-translational modifications and their heterogeneities, including the site-specific glycosylation, were analyzed by mass spectrometric peptide/glycopeptide mapping of trypsin-digested rhTM and precursor-ion scanning. Precursor-ion scanning was successful in the detection of five glycopeptides. Four N-glycosylation sites and their site-specific carbohydrate heterogeneity were determined by their mass spectra. O-Glycosylation could be estimated on the basis of its mass spectrum. We were able to identify partial beta-hydroxylation on Asn324 and Asn439, and O-linked glucose on Ser287 from the peptide/glycopeptide map and their mass spectra. We demonstrated that a sequential analysis of LC-MS and LC-MS-MS are very useful for the structural analysis of O- and N-linked glycans, polypeptides, and post-translational modifications and their heterogeneities, including site-specific glycosylation in a glycoprotein. Our method can be applied to a glycoprotein in biological samples.

Amino Acid Sequence↗
PubMed2002

Simultaneous microanalysis of N-linked oligosaccharides in a glycoprotein using microbore graphitized carbon column liquid chromatography-mass spectrometry.

We previously reported that graphitized carbon column liquid chromatography-mass spectrometry (GCC-LC-MS) is very useful for the structural analysis of carbohydrates in a glycoprotein. In this study, GCC-LC-MS was adapted for the simultaneous microanalysis of oligosaccharides. A variety of oligosaccharide alditols prepared from fetuin, ribonuclease B, and recombinant human erythropoietin were used as model oligosaccharides. The use of microbore GCC-LC-MS was found to be successful for rapid, sensitive, and simultaneous analysis of high-mannose-type, desialylated fucosyl complex-type, sialylated complex-type, and sialylated fucosyl complex-type oligosaccharide alditols. Furthermore, we demonstrate that this method is applicable to the analysis of carbohydrate heterogeneity in a glycoprotein that possesses diverse oligosaccharides. Microbore GCC-LC-MS was able to characterize high-mannose-type, hybrid-type, and complex-type oligosaccharides in tissue plasminogen activator produced from human melanoma cells in a single analysis.

Carbohydrate Sequence↗
PubMed2002

Usefulness of sugar mapping by liquid chromatography/mass spectrometry in comparability assessments of glycoprotein products.

We have previously reported that sugar-mapping by liquid chromatography/mass spectrometry (LC/MS) equipped with a graphitized carbon column (GCC) can be useful for structural analysis of carbohydrates in a glycoprotein. In this paper, we evaluated sugar-mapping with regard to its use in comparability assessment of glycoprotein products. Erythropoietins (EPO) produced from three different sources were chosen as models of the closely related glycoprotein products. The two-dimensional displays of sugar maps drawn by LC/MS with GCC clearly showed the differences in carbohydrate heterogeneity with regard to sialylation, acetylation, and sulphation patterns among three EPOs. Exoglycosidase digestion followed by sugar-mapping provided information regarding the structure of characteristic carbohydrates in each EPO. These results demonstrate that LC/MS with GCC can reveal the details of carbohydrate heterogeneity in order to distinguish between closely related glycoprotein products. Our method can thus be useful in comparability assessments of therapeutic glycoproteins.

Animals↗
PubMed2002

Usefulness of glycopeptide mapping by liquid chromatography/mass spectrometry in comparability assessment of glycoprotein products.

We previously reported on glycopeptide mapping of erythropoietin (EPO) by liquid chromatography/mass spectrometry (LC/MS). Using this method, glycopeptides in proteolytic digestion can be eluted before peptides, and are further separated on the basis of the carbohydrate structure. The detailed glycosylation at each glycosylation site can be elucidated based on mass chromatography and mass spectroscopy. In this study, we evaluated glycopeptide mapping with regard to its use in comparability assessment of glycoprotein products possessing multiple glycosylation sites. Models of closely related glycoprotein products used in this study are EPOs produced from three different sources. We previously reported that there are differences in the carbohydrate heterogeneity of these EPOs with regard to sialylation, acetylation, and sulphation patterns, using sugar mapping by LC/MS. In this paper, we demonstrated that glycopeptide mapping can distinguish site-specific glycosylation among these three EPOs and reveal the differences in acetylation, sialylation, and sulphation at each glycosylation site in one analysis. Our method can thus be useful in comparability assessment of therapeutic glycoproteins in terms of glycosylation.

Animals↗
PubMed2002

[Study on evaluating methods for the quality control of glycoprotein products. (IV)--erythropoietin products. Part 4].

We have previously reported on peptide mapping of recombinant human erythropoietin (rhEPO) by liquid chromatography/mass spectrometry (LC/MS). Using this method, both peptides and glycosylation at each glycosylation site can be elucidated based on the mass chromatogram and mass spectrum. In this study, we evaluated the mass spectrometric peptide mapping with regard to its use in comparability assessment of both protein parts and carbohydrates parts in glycoprotein products. Models of closely related glycoprotein products used in this study are rhEPOs produced from three different sources. We demonstrated that the mass spectrometric peptide mapping can elucidate the identity of protein part, and differences in site-specific carbohydrates heterogeneity due to acetylation and sulfation among the three rhEPOs. Our method can thus be useful in comparability assessment of therapeutic glycoproteins.

Binding Sites↗