Language：English   Publishing type：Research paper (scientific journal)  

Aberrant glycosylation is a prominent feature of cancer, that can be used as targets to improve the existing cancer biomarkers, and help to assess metastasis risks, and therapeutic effects. We developed a targeted O-glycoproteomics method using serum specimens, and evaluated its utility in identifying advanced colorectal cancer (CRC) markers. To this end, we combined consecutive lectin affinity purification using Maclura pomifera lectin (MPL), jacalin, and Sambucus nigra lectin, which have affinities for the following O-glycans, that have received attention as cancer-related antigens, Tn (GalNAc-Ser/Thr), Sialyl Tn (Siaα2-6GalNAc-Ser/Thr), T (Galβ1-3GalNAc-Ser/Thr), Sialyl T (Siaα2-3Galβ1-GalNAc-Ser/Thr), and di-Sialyl T (Siaα2-3Galβ1-3[Siaα2-6] GalNAc-Ser/Thr), with a unique O-glycoproteomics approach. A total of 2,068 O-glycoforms derived from 265 proteins were identified in healthy individuals and patients with advanced CRC, of which 44 CRC-specific O-glycoforms were extracted. Particularly, five glycoproteins with T, Sialyl T, and di-Sialyl T antigens in specific peptide regions were evaluated quantitatively and statistically. We found that fibulin-2 (FBLN2) (aa330-349)/T antigen (area under the curve [AUC] = 0.92); macrophage colony-stimulating factor 1 (CSF1) (aa370-395)/(T + di-Sialyl T) (AUC = 0.94); macrophage mannose receptor 1 (MRC1) (aa1083-1101 and aa1215-1229)/T (AUC = 0.96 and 0.99); fibrinogen alpha chain (FGA) (aa354-367, aa511-527 and aa559-573)/Sialyl T (AUC = 0.98, 0.90 and 0.94); and complement component C7 (C7) (aa692-701)/di-Sialyl T (AUC = 1.00), can have high diagnostic efficacy to strategically predict advanced CRC groups. Hence, they could be promising markers for detection of advanced CRC, and provide new clinical test indicators along with lectins, such as MPL and jacalin. Our O-glycoproteomics platform provides a novel tool and resource, for researchers and clinicians seeking to better understand and treat advanced CRC.

DOI： 10.3389/fonc.2023.1104936

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Appropriate nomenclature for all pharmaceutical substances is important for clinical development, licensing, prescribing, pharmacovigilance, and identification of counterfeits. Nonproprietary names that are unique and globally recognized for all pharmaceutical substances are assigned by the International Nonproprietary Names (INN) Programme of the World Health Organization (WHO). In 1991, the INN Programme implemented the first nomenclature scheme for monoclonal antibodies. To accompany biotechnological development, this nomenclature scheme has evolved over the years; however, since the scheme was introduced, all pharmacological substances that contained an immunoglobulin variable domain were coined with the stem -mab. To date, there are 879 INN with the stem -mab. Owing to this high number of names ending in -mab, devising new and distinguishable INN has become a challenge. The WHO INN Expert Group therefore decided to revise the system to ease this situation. The revised system was approved and adopted by the WHO at the 73rd INN Consultation held in October 2021, and the radical decision was made to discontinue the use of the well-known stem -mab in naming new antibody-based drugs and going forward, to replace it with four new stems: -tug, -bart, -mig, and -ment.

DOI： 10.1080/19420862.2022.2075078

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Language：English   Publishing type：Research paper (scientific journal)  

The generation of mature synaptic structures using neurons differentiated from human-induced pluripotent stem cells (hiPSC-neurons) is expected to be applied to physiological studies of synapses in human cells and to pathological studies of diseases that cause abnormal synaptic function. Although it has been reported that synapses themselves change from an immature to a mature state as neurons mature, there are few reports that clearly show when and how human stem cell-derived neurons change to mature synaptic structures. This study was designed to elucidate the synapse formation process of hiPSC-neurons. We propagated hiPSC-derived neural progenitor cells (hiPSC-NPCs) that expressed localized markers of the ventral hindbrain as neurospheres by dual SMAD inhibition and then differentiated them into hiPSC-neurons in vitro. After 49 days of in vitro differentiation, hiPSC-neurons significantly expressed pre- and postsynaptic markers at both the transcript and protein levels. However, the expression of postsynaptic markers was lower than in normal human or normal rat brain tissues, and immunostaining analysis showed that it was relatively modest and was lower than that of presynaptic markers and that its localization in synaptic structures was insufficient. Neurophysiological analysis using a microelectrode array also revealed that no synaptic activity was generated on hiPSC-neurons at 49 days of differentiation. Analysis of subtype markers by immunostaining revealed that most hiPSC-neurons expressed vesicular glutamate transporter 2 (VGLUT2). The presence or absence of NGF, which is required for the survival of cholinergic neurons, had no effect on their cell fractionation. These results suggest that during the synaptogenesis of hiPSC-neurons, the formation of presynaptic structures is not the only requirement for the formation of postsynaptic structures and that the mRNA expression of postsynaptic markers does not correlate with the formation of their mature structures. Technically, we also confirmed a certain level of robustness and reproducibility of our neuronal differentiation method in a multicenter setting, which will be helpful for future research. Synapse formation with mature postsynaptic structures will remain an interesting issue for stem cell-derived neurons, and the present method can be used to obtain early and stable quality neuronal cultures from hiPSC-NPCs.

DOI： 10.1186/s13041-021-00851-1

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Language：English   Publishing type：Research paper (scientific journal)  

N-glycosylation of glycoproteins, a major post-translational modification, plays a crucial role in various biological phenomena. In central nervous systems, N-glycosylation is thought to be associated with differentiation and regeneration; however, the state and role of N-glycosylation in neuronal differentiation remain unclear. Here, we conducted sequential LC/MS/MS analyses of tryptic digest, enriched glycopeptides, and deglycosylated peptides of proteins derived from human-induced pluripotent stem cells (iPSCs) and iPSC-derived neuronal cells, which were used as a model of neuronal differentiation. We demonstrate that the production profiles of many glycoproteins and their glycoforms were altered during neuronal differentiation. Particularly, the levels of glycoproteins modified with an N-glycan, consisting of five N-acetylhexosamines, three hexoses, and a fucose (HN5H3F), increased in dopaminergic neuron-rich cells (DAs). The N-glycan was deduced to be a fucosylated and bisected biantennary glycan based on product ion spectra. Interestingly, the HN5H3F-modified proteins were predicted to be functionally involved in neural cell adhesion, axon guidance, and the semaphorin-plexin signaling pathway, and protein modifications were site-selective and DA-selective regardless of protein production levels. Our integrated method for glycoproteome analysis and resultant profiles of glycoproteins and their glycoforms provide valuable information for further understanding the role of N-glycosylation in neuronal differentiation and neural regeneration.

DOI： 10.1038/s41598-021-90102-z

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Language：English   Publishing type：Research paper (scientific journal)  

We developed two human-induced pluripotent stem cell (hiPSC)/human embryonic stem cell (hESC)-specific glycan-recognizing mouse antibodies, R-10G and R-17F, using the Tic (JCRB1331) hiPSC line as an antigen. R-10G recognizes a low-sulfate keratan sulfate, and R-17F recognizes lacto-N-fucopentaose-1. To evaluate the general characteristics of stem cell glycans, we investigated the hiPSC line 201B7 (HPS0063), a prototype iPSC line. Using an R-10G affinity column, an R-10G-binding protein was isolated from 201B7 cells. The protein yielded a single but very broad band from 480 to 1236 kDa by blue native gel electrophoresis. After trypsin digestion, the protein was identified as podocalyxin by liquid chromatography/mass spectrometry. According to Western blotting, the protein reacted with R-10G and R-17F. The R-10G-positive band was resistant to digestion with glycan-degrading enzymes, including peptide N-glycanase, but the intensity of the band was decreased significantly by digestion with keratanase, keratanase II, and endo-β-galactosidase, suggesting the R-10G epitope to be a keratan sulfate. These results suggest that keratan sulfate-type epitopes are shared by hiPSCs. However, the keratan sulfate from 201B7 cells contained a polylactosamine disaccharide unit (Galβ1-4GlcNAc) at a significant frequency, whereas that from Tic cells consisted mostly of keratan sulfate disaccharide units (Galβ1-4GlcNAc(6S)). In addition, the abundance of the R-10G epitope was significantly lower in 201B7 cells than in Tic cells.

DOI： 10.3390/biom11040508

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Language：English   Publishing type：Research paper (scientific journal)  

In the mammalian nervous system, protein N-glycosylation plays an important role in neuronal physiology. In this study, we performed a comprehensive N-glycosylation analysis of mouse GluA1, one of the major subunits of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate type glutamate receptor, which possesses six potential N-glycosylation sites in the N-terminal domain. By mass spectrometry-based analysis, we identified the N-glycoforms and semiquantitatively determined the site-specific N-glycosylation occupancy of GluA1. In addition, only the N401-glycosylation site demonstrated incomplete N-glycosylation occupancy. Therefore, we generated a peptide antibody that specifically detects the N401-glycan-free form to precisely quantify N401-glycosylation occupancy. Using this antibody, we clarified that N401 occupancy varies between cell types and increases in an age-dependent manner in mouse forebrains. To address the regulatory mechanism of N401-glycosylation, binding proteins of GluA1 around the N401 site were screened. HSP70 family proteins, including Bip, were identified as candidates. Bip has been known as a molecular chaperone that plays a key role in protein folding in the ER (endoplasmic reticulum). To examine the involvement of Bip in N401-glycosylation, the effect of Bip over-expression on N401 occupancy was evaluated in HEK293T cells, and the results demonstrated Bip increases the N401 glycan-free form by mediating selective prolongation of its protein half-life. Taken together, we propose that the N401-glycosite of GluA1 receives a unique control of modification, and we also propose a novel N-glycosylation occupancy regulatory mechanism by Bip that might be associated with α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors function in the brain.

DOI： 10.1111/jnc.14964

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

A class of glycoproteins such as carcinoembryonic antigen (CEA)/CEA-related cell adhesion molecule 1(CEACAM1), CD26 (DPPIV), and mac-2 binding protein (Mac-2BP) harbor tumor-associated glycans in colorectal cancer. In this study, we identified type II transmembrane mosaic serine protease large-form (MSPL) and its splice variant transmembrane protease serine 13 (TMPRSS13) as ligands of Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) on the colorectal cancer cells. DC-SIGN is a C-type lectin expressed on dendritic cells, serves as a pattern recognition receptor for numerous pathogens such as human immunodeficiency virus (HIV) and M. tuberculosis. DC-SIGN recognizes these glycoproteins in a Ca2+ dependent manner. Meanwhile, we found that MSPL proteolytically cleaves DC-SIGN in addition to the above glycan-mediated recognition. DC-SIGN was degraded more efficiently by MSPL when treated with ethylenediaminetetraacetic acid (EDTA), suggesting that glycan-dependent interaction of the two molecules partially blocked DC-SIGN degradation. Our findings uncovered a dual recognition system between DC-SIGN and MSPL/TMPRSS13, providing new insight into the mechanism underlying colorectal tumor microenvironment.

DOI： 10.3390/app10082687

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Informa UK Limited  

Glycosylation of the conserved asparagine residue in each heavy chain of IgG in the CH2 domain is known as N-glycosylation. It is one of the most common post-translational modifications and important critical quality attributes of monoclonal antibody (mAb) therapeutics. Various studies have demonstrated the effects of the Fc N-glycosylation on safety, Fc effector functions, and pharmacokinetics, both dependent and independent of neonatal Fc receptor (FcRn) pathway. However, separation of various glycoforms to investigate the biological and functional relevance of glycosylation is a major challenge, and existing studies often discuss the overall impact of N-glycans, without considering the individual contributions of each glycoform when evaluating mAbs with highly heterogeneous distributions. In this study, chemoenzymatic glycoengineering incorporating an endo-β-N-acetylglucosaminidase (ENGase) EndoS2 and its mutant with transglycosylation activity was used to generate mAb glycoforms with highly homogeneous and well-defined N-glycans to better understand and precisely evaluate the effect of each N-glycan structure on Fc effector functions and protein stability. We demonstrated that the core fucosylation, non-reducing terminal galactosylation, sialylation, and mannosylation of IgG1 mAb N-glycans impact not only on FcγRIIIa binding, antibody-dependent cell-mediated cytotoxicity, and C1q binding, but also FcRn binding, thermal stability and propensity for protein aggregation.

DOI： 10.1080/19420862.2018.1551044

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Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Glycosylation is highly susceptible to changes of the physiological conditions, and accordingly, is a potential biomarker associated with several diseases and/or longevity. Semi-supercentenarians (SSCs; older than 105 years) are thought to be a model of human longevity. Thus, we performed glycoproteomics using plasma samples of SSCs, and identified proteins and conjugated N-glycans that are characteristic of extreme human longevity. METHODS: Plasma proteins from Japanese semi-supercentenarians (SSCs, 106-109 years), aged controls (70-88 years), and young controls (20-38 years) were analysed by using lectin microarrays and liquid chromatography/mass spectrometry (LC/MS). Peak area ratios of glycopeptides to corresponding normalising peptides were subjected to orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, plasma levels of clinical biomarkers were measured. RESULTS: We found two lectins such as Phaseolus vulgaris, and Erythrina cristagalli (ECA), of which protein binding were characteristically increased in SSCs. Peak area ratios of ECA-enriched glycopeptides were successfully discriminated between SSCs and controls using OPLS-DA, and indicated that tri-antennary and sialylated N-glycans of haptoglobin at Asn207 and Asn211 sites were characterized in SSCs. Sialylated glycans of haptoglobin are a potential biomarker of several diseases, such as hepatocellular carcinoma, liver cirrhosis, and IgA-nephritis. However, the SSCs analysed here did not suffer from these diseases. CONCLUSIONS: Tri-antennary and sialylated N-glycans on haptoglobin at the Asn207 and Asn211 sites were abundant in SSCs and characteristic of extreme human longevity. GENERAL SIGNIFICANCE: We found abundant glycans in SSCs, which may be associated with human longevity.

DOI： 10.1016/j.bbagen.2018.03.025

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for the Study of Xenobiotics  

This study was undertaken to evaluate the performance of anti-drug antibody (ADA) assays constructed by each participating company using common samples including ADA, drug and human serum. The ADA assays constructed by each company showed good sensitivity and precision for evaluation of ADA. Cut points for screening and confirmatory assays and assay selectivity were determined by various calculation methods. In evaluations of blind ADA samples, nearly similar results were obtained by the study companies in determinations of whether samples were positive or negative except at the lowest sample concentration (5 ng/mL). In measurement of drug tolerance, for almost samples containing ADA and drugs, more positive results were obtained in assays using acid dissociation compared to those without acid dissociation. Overall, the performance of ADA assays constructed by the 10 companies participating in this study was acceptable in terms of sensitivity and reproducibility for detection and evaluation of immunogenicity in both patients and healthy subjects. On the other hand, based on results for samples containing ADA and drugs, validity of results for ADA assays conducted without acid dissociation was less meaningful and more difficult to evaluate. Thus, acid dissociation was confirmed to be useful for improving drug tolerance.

DOI： 10.1016/j.dmpk.2018.03.002

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Fc-receptors for immunoglobulin G (FcγRs) mediate a variety of effector and regulatory mechanisms in the immune system. N-glycosylation of FcγRs critically affects their functions which is well exemplified by antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis mediated by homologous FcγRIIIa and FcγRIIIb, respectively. Although several reports describe N-glycosylation profiles of recombinant FcγRIII glycoproteins, much remains unknown regarding their native glycoforms. Here we performed site-specific N-glycosylation profiling of a soluble form of FcγRIIIb purified from human serum based on mass spectrometric analysis. Our data indicate a distinct and common tendency of the glycoforms exhibited at each N-glycosylation site between the native and the previously reported recombinant FcγRIII glycoproteins. Among the six N-glycosylation sites of serum soluble FcγRIIIb, Asn45 was shown to be exclusively occupied by high-mannose-type oligosaccharides, whereas the remaining sites were solely modified by the complex-type oligosaccharides with sialic acid and fucose residues. The results of our endogenous FcγRIII glycoform analyses are important for the optimization of therapeutic antibody efficacy.

DOI： 10.1038/s41598-018-21145-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Recently, we established two mouse monoclonal antibodies (R-10G and R-17F). The R-17F antibody (IgG1 subtype) exhibited a strong cytotoxic effect on hiPS/ES cells. The R-17F antigen isolated from a total lipid extract of hiPS (Tic) cells was identified as LNFP I (Fuc alpha 1-2Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc). In the present study, R-17F binding proteins were isolated from hiPS (Tic) cell lysates with an affinity column of R-17F. They gave one major R-17F positive band around 250 kDa, and several minor bands between 150 kDa and 25 kDa. The former band was identified as podocalyxin by LC/MS/MS after SDS-PAGE. Hapten inhibition studies on R-17F binding to R-17F column-purified proteins with various synthetic oligosaccharides revealed that the blood group H type 1 triaose structure (Fuc alpha 1-2Gal beta 1-3GlcNAc) was the predominant epitope on all the R-17F binding proteins. These bands disappeared completely on digestion with alpha 1-2 fucosidase, but not with alpha 1-3/4 fucosidase. Upon PNGase F digestion, the R-17F positive band around and above 250 kDa did not show any change, while the minor bands between 150 kDa and 25 kDa disappeared completely, suggesting that the epitope is expressed on N-glycans in the latter and probably on O-glycans in the former. These results, together with those obtained in our previous studies on R-10G (Kawabe et al. Glycobiology, 23, 322-336 (2013)), indicated that both R-10G and R-17F epitopes are carried on the same podocalyxin molecule. The R-17F epitopes on these glycoproteins expressed on hiPS cells could be associated with the molecular mechanism underlying the carbohydrate-mediated cytotoxic activity of R-17F.

DOI： 10.1007/s10719-016-9710-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

The cytoplasmic peptide: N-glycanase (Ngly1 in mammals) is a de-N-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the NGLY1 gene exhibited severe systemic symptoms (NGLY1-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed Ngly1-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-beta N--acetylglucosaminidase (Engase), which is another de-N-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the Ngly1-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of Ngly1-deficient mice. Viable Ngly1deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of NGLY1-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of Engase in the C57BL/6 and ICR mixed background. The defects observed in Ngly1/Engase-deficient mice (C57BL/6 background) and Ngly1-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an NGLY1-deficiency. These observations strongly suggest that the Ngly1-or Ngly1/Engase-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the NGLY1-deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder.

DOI： 10.1371/journal.pgen.1006696

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The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.

DOI： 10.1007/s10719-015-9625-3

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1038/ncomms11205

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Other Link： http://www.nature.com/articles/ncomms11205

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Some aberrant N-glycosylations are being used as tumor markers, and glycoproteomics is expected to provide novel diagnosis markers and targets of drug developments. However, one has trouble in mass spectrometric glycoproteomics of membrane fraction because of lower intensity of glycopeptides in the existence of surfactants. Previously, we developed a glycopeptide enrichment method by acetone precipitation, and it was successfully applied to human serum glycoproteomics. In this study, we confirmed that this method is useful to remove the surfactants and applicable to membrane glycoproteomics. The glycoproteomic approach to the human fetal lung fibroblasts membrane fraction resulted in the identification of over 272 glycoforms on 63 sites of the 44 glycoproteins. According to the existing databases, the structural features on 41 sites are previously unreported. The most frequently occurring forms at N-glycosylation site were high-mannose type containing nine mannose residues (M9) and monosialo-fucosylated biantennary oligosaccharides. Several unexpected N-glycans, such as fucosylated complex-type and fucosylated high-mannose and/or fucosylated pauci-mannose types were found in ER and lysosome proteins. Our method provides new insights into transport, biosynthesis, and degradation of glycoproteins.

DOI： 10.1002/pmic.201500003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Human natural killer-1 (HNK-1) carbohydrate (HSO3-3GlcA beta 1-3Gal beta 1-4GlcNAc-R) is highly expressed in the brain and required for learning and neural plasticity. We previously demonstrated that expression of the HNK-1 epitope is mostly abolished in knockout mice for GlcAT-P (B3gat1), a major glucuronyltransferase required for HNK-1 biosynthesis, but remained in specific regions such as perineuronal nets (PNNs) in these mutant mice. Considering PNNs are mainly composed of chondroitin sulfate proteoglycans (CSPGs) and regulate neural plasticity, GlcAT-P-independent expression of HNK-1 in PNNs is suggested to play a role in neural plasticity. However, the function, structure, carrier glycoprotein and biosynthetic pathway for GlcAT-P-irrelevant HNK-1 epitope remain unclear. In this study, we identified a unique HNK-1 structure on aggrecan in PNNs. To determine the biosynthetic pathway for the novel HNK-1, we generated knockout mice for GlcAT-S (B3gat2), the other glucuronyltransferase required for HNK-1 biosynthesis. However, GlcAT-P and GlcAT-S double-knockout mice did not exhibit reduced HNK-1 expression compared with single GlcAT-P-knockout mice, indicating an unusual biosynthetic pathway for the HNK-1 epitope in PNNs. Aggrecan was purified from cultured cells in which GlcAT-P and -S are not expressed and we determined the structure of the novel HNK-1 epitope using liquid chromatography/mass spectrometry (LC/MS) as a sulfated linkage region of glycosaminoglycans (GAGs), HSO3-GlcA-Gal-Gal-Xyl-R. Taken together, we propose a hypothetical model where GlcAT-I, the sole glucuronyltransferase required for synthesis of the GAG linkage, is also responsible for biosynthesis of the novel HNK-1 on aggrecan. These results could lead to discovery of new roles of the HNK-1 epitope in neural plasticity.

DOI： 10.1371/journal.pone.0144560

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera (R)). The anti-CD20mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO- and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.

DOI： 10.1080/19420862.2015.1078054

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Language：Japanese   Publisher：(一財)医薬品医療機器レギュラトリーサイエンス財団  

第十六改正日局第二追補ヘパリン定量法に従い測定を実施し、第IIa因子液、アンチトロンビン液について、それぞれ日局記載の濃度の1〜2倍、0.125〜1倍を用い測定し、これらの試験濃度を限定しない試験法の設定について検討した。現在流通しているトロンビン試薬、アンチトロンビン試薬中から第十六改正日局第二追補ヘパリン定量法で良好な定量が可能な試薬を選定した。また、トロンビンおよびアンチトロンビンの濃度を限定せず、空試験液の反応液の吸光度が2.0以下、ヘパリン標準液S4の反応液の吸光度が0.2以上1.0以下と設定することで、良好な試験が可能であることが明らかとなった。

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Language：Japanese   Publisher：日本生殖免疫学会  

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Language：English   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.jri.2015.09.034

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry {\&} Molecular Biology ({ASBMB})  

DOI： 10.1074/jbc.m115.670901

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Ts4, an anti-sperm auto-monoclonal antibody, possesses immunoreactivity to the acrosomal region of mouse epididymal spermatozoa. In addition, the mAb shows specific immunoreactivity to reproduction-related regions such as testicular germ cells and early embryo. Our qualitative study previously showed that the antigen epitope for Ts4 contained a N-linked common oligosaccharide (OS) chain on testicular glycoproteins as determined by Western blotting for testicular glycoproteins after treatment with several glycohydrolases. Since the distribution of the Ts4-epitope is unique, the OS chain in Ts4-epitope may have role(s) in the reproductive process. The aim of this study was to clarify the molecular structure of the Ts4-epitope, particularly its OS moiety. Using Ts4 immunoprecipitation combined with liquid chromatography and multiple-stage mass spectrometry, the candidate carbohydrate structure in the Ts4-epitope is proposed to be N-linked fucosylated agalacto-biantennary with bisecting N-acetylglucosamine (GlcNAc) or with N-acetylgalactosamine-GlcNAc motif. Further binding analyses using various lectins against the mouse testicular Ts4-immunoprecipitants revealed that Phaseolus vulgaris erythroagglutinin and Pisum sativum agglutinin showed positive staining of the bands corresponding to Ts4 reactive proteins. Moreover, the immunoreactivity of Ts4 against the testicular extract was completely abrogated after digestion with beta-N-acetylglucosaminidase. These results show that the Ts4-epitope contains agalacto-biantennary N-glycan with bisecting GlcNAc carrying fucose residues.

DOI： 10.1371/journal.pone.0133784

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Language：Japanese   Publisher：(公社)日本薬学会  

抗体医薬品は、抗原の中和や、fragment crystallizable(Fc)受容体の活性化を介した細胞傷害活性などにより、薬理作用を発揮する。ヒト蛋白質に対して特異的に作用する抗体医薬品は、しばしば動物の相同蛋白質との反応性を欠き、非臨床試験での有効性・安全性評価が難しくなる。非臨床試験系の妥当性を評価する際には、Fc受容体についても十分に考慮する必要がある。Fc受容体との関連を中心に、次世代抗体医薬品の非臨床評価の課題について考察した。

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2015&ichushi_jid=J01459&link_issn=&doc_id=20150715300003&doc_link_id=130005085757&url=http%3A%2F%2Fci.nii.ac.jp%2Fnaid%2F130005085757&type=CiNii&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00003_3.gif

Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

Many monoclonal antibodies have been developed for therapy over the last 2 decades. In the development of therapeutic antibodies, the preclinical assessment of an antibody's biodistribution is important for the prediction of the antibody's efficacy and safety. For imaging analyses of such biodistributions, radioisotope (RI) labeling and fluorescence labeling methods are typically used, but the resulting data are limited because these methods cannot distinguish breakdown products from intact antibodies. To resolve this problem, we developed a novel method using fluorescent resonance energy transfer (FRET)-type labeling and a spectral unmixing tool. With FRET-type labeling (labeling with 2 species of fluorophore), different fluorescence properties of labeled intact antibodies and their breakdown products (the hydrolyzed/digested type of breakdown products) are made visible. With the spectral unmixing tool, the fluorescence of a solution containing the intact antibody and its breakdown products could be unmixed in proportion to their contents. Moreover, when labeled antibodies that targeted either human epidermal growth factor receptor-2 or epidermal growth factor receptor were injected into nude mice implanted subcutaneously with tumor cells, the accumulation of the injected labeled antibodies and their breakdown products in the tumor could be separately analyzed by both whole-mouse imaging and a tumor homogenate analysis. These results suggest that our method using FRET-type labeling and a spectral unmixing tool could be useful in distinguishing breakdown products from intact antibodies.

DOI： 10.1080/19420862.2015.1038683

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Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing (15)N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a (15)N-enrichment ratio of approximately 80%. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies.

DOI： 10.1007/s10858-015-9930-y

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KEY MESSAGE: We successfully developed a method for metabolic isotope labeling of recombinant proteins produced in transgenic tobacco. This enabled assessment of structural integrity of plant-derived therapeutic antibodies by NMR analysis. A variety of expression vehicles have been developed for the production of promising biologics, including plants, fungi, bacteria, insects, and mammals. Glycoprotein biologics often experience altered folding and post-translational modifications that are typified by variant glycosylation patterns. These differences can dramatically affect their efficacy, as exemplified by therapeutic antibodies. However, it is generally difficult to validate the structural integrity of biologics produced using different expression vehicles. To address this issue, we have developed and applied a stable-isotope-assisted nuclear magnetic resonance (NMR) spectroscopy method for the conformational characterization of recombinant antibodies produced in plants. Nicotiana benthamiana used as a vehicle for the production of recombinant immunoglobulin G (IgG) was grown in a (15)N-enriched plant growth medium. The Fc fragment derived from the (15)N-labeled antibody thus prepared was subjected to heteronuclear two-dimensional (2D) NMR measurements. This approach enabled assessment of the structural integrity of the plant-derived therapeutic antibodies by comparing their NMR spectral properties with those of an authentic IgG-Fc derived from mammalian cells.

DOI： 10.1007/s00299-015-1757-1

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Language：Japanese   Publisher：(公社)日本薬学会  

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2015&ichushi_jid=J01316&link_issn=&doc_id=20150508260001&doc_link_id=10.14894%2Ffaruawpsj.51.5_403&url=https%3A%2F%2Fdoi.org%2F10.14894%2Ffaruawpsj.51.5_403&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

Language：Japanese   Publisher：(株)じほう  

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DOI： 10.1371/journal.pone.0142645

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β-Galactoside α2,6-sialyltranferase 1 (ST6GAL1) catalyzes the addition of terminal α2,6-sialylation to N-glycans. Increased expression of ST6GAL1 has been reported in diverse carcinomas and highly correlates with tumor progression. Here, we report that St6gal1 transcription and α2,6-sialylated N-glycans are up-regulated during TGF-β-induced epithelial-mesenchymal transition (EMT) in GE11 cells, requiring the Sp1 element within the St6gal1 promoter. Knockdown of St6gal1 strongly suppressed TGF-β-induced EMT with a concomitant increase in E-cadherin expression, a major determinant of epithelial cell adherens junctions. Conversely, overexpression of ST6GAL1 increased the turnover of cell surface E-cadherin and promoted TGF-β-induced EMT. Overexpressing β-galactoside α2,3-sialyltranferase 4 had little influence on EMT, indicating specificity for α2,6-sialylation. The basal mesenchymal phenotype of MDA-MB-231 human breast cancer cells was partially reversed by ST6GAL1 silencing. Moreover, ST6GAL1 knockdown inhibited the phosphorylation of Akt, but not Smad2, suggesting that ST6GAL1 contributes to EMT through a non-Smad signaling pathway. Taken together, our data indicate that ST6GAL1 promotes TGF-β-dependent EMT as well as maintenance of the mesenchymal state by growth signaling, providing a plausible mechanism whereby up-regulated ST6GAL1 may promote malignant progression.

DOI： 10.1074/jbc.M114.593392

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Language：English   Publisher：OXFORD UNIV PRESS INC  

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Language：Japanese   Publisher：(一財)医薬品医療機器レギュラトリーサイエンス財団  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Background:N-Glycan 2,6-sialylation regulates the cell surface residency of an anti-apoptotic molecule, platelet endothelial cell adhesion molecule (PECAM). Results: An 2,6-sialylated oligosaccharide inhibited the homophilic PECAM interaction and a cluster-type 2,6-sialyl N-glycan probe bound to PECAM-immobilized beads. Conclusion: PECAM is a weak sialic acid binding lectin. Significance: There is a possibility of using a glycan-based method to modulate angiogenesis.
The luminal sides of vascular endothelial cells are heavily covered with a so-called glycocalyx, but the precise role of the endothelial glycocalyx remains unclear. Our previous study showed that N-glycan 2,6-sialylation regulates the cell surface residency of an anti-apoptotic molecule, platelet endothelial cell adhesion molecule (PECAM), as well as the sensitivity of endothelial cells toward apoptotic stimuli. As PECAM itself was shown to be modified with biantennary N-glycans having 2,6-sialic acid, we expected that PECAM would possess lectin-like activity toward 2,6-sialic acid to ensure its homophilic interaction. To verify this, a series of oligosaccharides were initially added to observe their inhibitory effects on the homophilic PECAM interaction in vitro. We found that a longer 2,6-sialylated oligosaccharide exhibited strong inhibitory activity. Furthermore, we found that a cluster-type 2,6-sialyl N-glycan probe specifically bound to PECAM-immobilized beads. Moreover, the addition of the 2,6-sialylated oligosaccharide to endothelial cells enhanced the internalization of PECAM as well as the sensitivity to apoptotic stimuli. Collectively, these findings suggest that PECAM is a sialic acid binding lectin and that this binding property supports endothelial cell survival. Notably, our findings that 2,6-sialylated glycans influenced the susceptibility to endothelial cell apoptosis shed light on the possibility of using a glycan-based method to modulate angiogenesis.

DOI： 10.1074/jbc.M114.563585

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Recently, the Golgi phosphoprotein 3 (GOLPH3) and its yeast homolog Vps74p have been characterized as essential for the Golgi localization of glycosyltransferase in yeast. GOLPH3 has been identified as a new oncogene that is commonly amplified in human cancers to modulate mammalian target of rapamycin signaling. However, the molecular mechanisms of the carcinogenic signaling pathway remain largely unclear. To investigate whether the expression of GOLPH3 was involved in the glycosylation processes in mammalian cells, and whether it affected cell behavior, we performed a loss-of-function study. Cell migration was suppressed in GOLPH3 knockdown (KD) cells, and the suppression was restored by a re-introduction of the GOLPH3 gene. HPLC and LC/MS analysis showed that the sialylation of N-glycans was specifically decreased in KD cells. The specific interaction between sialyltransferases and GOLPH3 was important for the sialylation. Furthermore, overexpression of alpha 2,6-sialyltransferase-I rescued cell migration and cellular signaling, both of which were blocked in GOLPH3 knockdown cells. These results are the first direct demonstration of the role of GOLPH3 in N-glycosylation to regulate cell biological functions.

DOI： 10.1074/jbc.M113.542688

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

LC/MS is commonly used for site-specific glycosylation analysis of glycoproteins in cells and tissues. A limitation of this technique is the difficulty in acquiring reliable mass spectra for glycopeptides, mainly due to their high heterogeneity and poor hydrophobicity. Here, we establish a versatile method for efficient glycopeptide enrichment to acquire reliable mass spectra. Several lines of evidence using model glycoproteins suggest that our method is based on the different solubility between non-glycosylated and glycosylated peptides in acetone. We also provide data showing that the acetone-precipitated glycopeptide enrichment was successful in acquiring a more comprehensive MS/MS data set for the various glycoforms of each glycopeptide in crude human serum. We propose that this method is a powerful tool for the acquisition of reliable mass spectra from trace amounts of glycopeptides and an alternative to lectin affinity enrichment.
Biological significance In this study, we established a versatile method for glycopeptide enrichment to acquire reliable mass spectra because of the limitation of conventional enrichment methods, such as lectin affinity chromatography. Our enrichment method is capable of isolating glycopeptides from complex peptide mixtures such as crude serum. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jprot.2014.02.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Antibody-dependent cellular cytotoxicity (ADCC) is one of the important mechanisms of action of the targeting of tumor cells by therapeutic monoclonal antibodies (mAbs). Among the human Fcc receptors (Fc gamma Rs), Fc gamma RIIIa is well known as the only receptor expressed in natural killer (NK) cells, and it plays a pivotal role in ADCC by IgG1-subclass mAbs. In addition, the contributions of Fc gamma RIIa to mAb-mediated cytotoxicity have been reported. Fc gamma RIIa is expressed in myeloid effector cells including neutrophils and macrophages, and it is involved in the activation of these effector cells. However, the measurement of the cytotoxicity via Fc gamma RIIa-expressing effector cells is complicated and inconvenient for the characterization of therapeutic mAbs. Here we report the development of a cell-based assay using a human Fc gamma RIIa-expressing reporter cell line. The Fc gamma RIIa reporter cell assay was able to estimate the activation of Fc gamma RIIa by antigen-bound mAbs by a very simple method in vitro. The usefulness of this assay for evaluating the activity of mAbs with different abilities to activate Fc gamma RIIa was confirmed by the examples including the comparison of the activity of the anti-CD20 mAb rituximab and its Fc-engineered variants, and two anti-EGFR mAbs with different IgG subclasses, cetuximab (IgG1) and panitumumab (IgG2). We also applied this assay to the characterization of a force-oxidized mAb, and we observed that oxidation significantly decreased the Fc gamma RIIa activation by EGFR-bound cetuximab. These results suggest that our Fc gamma RIIa reporter assay is a promising tool for the characterization of therapeutic mAbs, including Fc-engineered mAbs, IgG2-subclass mAbs, and their product-related variants.

DOI： 10.1371/journal.pone.0095787

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

RATIONALEGlycan heterogeneity on recombinant human erythropoietin (rEPO) product is considered to be one of the critical quality attributes, and similarity tests of glycan heterogeneities are required in the manufacturing process changes and developments of biosimilars. A method for differentiating highly complex and diverse glycosylations is needed to evaluate comparability and biosimilarity among rEPO batches and products manufactured by different processes.
METHODSThe glycan heterogeneities of nine rEPO products (four innovator products and five biosimilar products) were distinguished by multivariate analysis (MVA) using the peak area ratios of each glycan to the total peak area of glycans in mass spectra obtained by liquid chromatography/mass spectrometry (LC/MS) of N-glycans from rEPOs.
RESULTSPrincipal component analysis (PCA) using glycan profiles obtained by LC/MS proved to be a useful method for differentiating glycan heterogeneities among nine rEPOs. Using PC values as indices, we were able to visualize and digitalize the glycan heterogeneities of each rEPO. The characteristic glycans of each rEPO were also successfully identified by orthogonal partial least-squares discrimination analysis (OPLS-DA), an MVA method, using the glycan profile data.
CONCLUSIONSPCA values were useful for evaluating the relative differences among the glycan heterogeneities of rEPOs. The characteristic glycans that contributed to the differentiation were also successfully identified by OPLS-DA. PCA and OPLS-DA based on mass spectrometric data are applicable for distinguishing glycan heterogeneities, which are virtually indistinguishable on rEPO products. Copyright (c) 2014 John Wiley & Sons, Ltd.

DOI： 10.1002/rcm.6858

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

The human natural killer-1 (HNK-1) carbohydrate comprising a sulfated trisaccharide (HSO3-3GlcA beta 1-3Gal beta 1-4GlcNAc-) is expressed on N-linked and O-mannose-linked glycans in the nervous system and involved in learning and memory functions. Although whole/core glycan structures and carrier glycoproteins for the N-linked HNK-1 epitope have been studied, carrier glycoproteins and the biosynthetic pathway of the O-mannose-linked HNK-1 epitope have not been fully characterized. Here, using mass spectrometric analyses, we identified the major carrier glycoprotein of the O-linked HNK-1 as phosphacan in developing mouse brains and determined the major O-glycan structures having the terminal HNK-1 epitope from partially purified phosphacan. The O-linked HNK-1 epitope on phosphacan almost disappeared due to the knockout of protein O-mannose beta 1,2-N-acetylglucosaminyltransferase 1, an N-acetylglucosaminyltransferase essential for O-mannose-linked glycan synthesis, indicating that the reducing terminal of the O-linked HNK-1 is mannose. We also showed that glucuronyltransferase-P (GlcAT-P) was involved in the biosynthesis of O-mannose-linked HNK-1 using the gene-deficient mice of GlcAT-P, one of the glucuronyltransferases for HNK-1 synthesis. Consistent with this result, we revealed that GlcAT-P specifically synthesized O-linked HNK-1 onto phosphacan using cultured cells. Furthermore, we characterized the as-yet-unknown epitope of the 6B4 monoclonal antibody (mAb), which was thought to recognize a unique phosphacan glycoform. The reactivity of the 6B4 mAb almost completely disappeared in GlcAT-P-deficient mice, and exogenously expressed phosphacan was selectively recognized by the 6B4 mAb when co-expressed with GlcAT-P, suggesting that the 6B4 mAb preferentially recognizes O-mannose-linked HNK-1 on phosphacan. This is the first study to show that 6B4 mAb-reactive O-mannose-linked HNK-1 in the brain is mainly carried by phosphacan.

DOI： 10.1093/glycob/cwt116

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

In-gel digestion followed by LC/MS/MS is widely used for the identification of trace amounts of proteins and for the site-specific glycosylation analysis of glycoproteins in cells and tissues. A major limitation of this technique is the difficulty in acquiring reliable mass spectra for peptides present in minute quantities and glycopeptides with high heterogeneity and poor hydrophobicity. It is considered that the SDS used in electrophoresis can interact with proteins noncovalently and impede the ionization of peptides/glycopeptides. In this study, we report an improved in-gel digestion method to acquire reliable mass spectra of a trace amount of peptides/glycopeptides. A key innovation of our improved method is the use of guanidine hydrochloride, which forms complexes with the residual SDS molecules in the sample. The precipitation and removal of SDS by addition of the guanidine hydrochloride was successful in improving the S/N of peptides/glycopeptides in mass spectra and acquiring a more comprehensive MS/MS data set for the various glycoforms of each glycopeptide.

DOI： 10.1002/pmic.201300332

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Language：Japanese   Publisher：(公社)日本薬剤学会  

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2014&ichushi_jid=J01463&link_issn=&doc_id=20131220340001&doc_link_id=10.14843%2Fjpstj.74.4&url=https%3A%2F%2Fdoi.org%2F10.14843%2Fjpstj.74.4&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

Language：Japanese   Publisher：(株)医薬ジャーナル社  

日本ではソマトロピン、エポエチンアルファ、およびフィルグラスチムバイオ後続品(バイオシミラー)が上市され、さらに現在インフリキシマブ後続品が承認申請中である。バイオシミラー販売で先行する欧州では、ジェネリック医薬品市場で見られるほどの大きなインパクトはないが、バイオシミラー製品の販売実績はゆっくりと伸びている。日本におけるバイオ後続品の今後を考える時、欧州での取り組みや動向が参考になると思われる。(著者抄録)

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DOI： 10.4172/2153-0637.1000116

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Language：Japanese   Publisher：Japanese Proteomics Society (Japan Human Proteome Organisation)  

DOI： 10.14889/jhupo.2014.0.119.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Heparin is a sulfated glycosaminoglycan (GAG), which contains N-acetylated or N-sulfated glucosamine (GlcN). Heparin, which is generally obtained from the healthy porcine intestines, is widely used as an anticoagulant during dialysis and treatments of thrombosis such as disseminated intravascular coagulation. Dermatan sulfate (DS) and chondroitin sulfate (CS), which are galactosamine (GalN)-containing GAGs, are major process-related impurities of heparin products. The varying DS and CS contents between heparin products can be responsible for the different anticoagulant activities of heparin. Therefore, a test to determine the concentrations of GalN-containing GAG is essential to ensure the quality and safety of heparin products. In this study, we developed a method for determination of relative content of GalN from GalN-containing GAG in heparin active pharmaceutical ingredients (APIs). The method validation and collaborative study with heparin manufacturers and suppliers showed that our method has enough specificity, sensitivity, linearity, repeatability, reproducibility, and recovery as the limiting test for GalN from GalN-containing GAGs. We believe that our method will be useful for ensuring quality, efficacy, and safety of pharmaceutical heparins. On July 30, 2010, the GalN limiting test based on our method was adopted in the heparin sodium monograph in the Japanese Pharmacopoeia. (C) 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biologicals.2013.06.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Heparin is used as an anticoagulant drug. The anticoagulation process is mainly caused by the interaction of heparin with antithrombin followed by inhibition of anticoagulant factor IIa and factor Xa. The anti-factor Ha and anti-factor Xa activities of heparin are critical for its anticoagulant effect; however, physicochemical methods that can reflect these activities have not been established. Thus, the measurements of anti-IIa and anti-Xa activities by biological assay are critical for the quality control of heparin products. Currently in the Japanese Pharmacopoeia UP), the activities of heparin sodium and heparin calcium are measured by an anti-Xa activity assay (anti-Xa assay), but anti-IIa activity is not measured. Here, we established an anti-IIa activity assay (anti-IIa assay) and an anti-Xa assay having good accuracy and precision. When samples having a relative activity of 0.8, 1.0 and 1.2 were measured by the established anti-IIa and anti-Xa assays in nine laboratories, good accuracy (100.0-102.8% and 101.6-102.8%, respectively), good intermediate precision (1.9-2.1% and 2.4-4.2%, respectively) and good reproducibility (4.0-4.8% and 3.6-6.4%, respectively) were obtained. The established anti-IIa and anti-Xa assays have similar protocols, and could be performed by a single person without a special machine. The established assays would be useful for quality control of heparin. (C) 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biologicals.2013.09.003

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The recent patent expirations of erythropoietin (EPO) have promoted the development of biosimilars. Two and one biosimilar EPO products were approved in 2007 in Europe and in 2010 in Japan, respectively. Glycosylation heterogeneity of EPO is very complex, and its pattern has a large impact on its in vivo activity. In this study, glycoform profilings of biosimilar and innovator EPO products were performed using LC/ESI-MS. Glycoforms of EPO were detected within the range of m/z 1700-3600 at the 10(+)-16(+) charge states. The charge-deconvoluted spectra showed complex glycoform mass profiles at 28,000-32,000 Da, and most of the observed peaks were assigned to the peptide (18,236 Da)+glycans with the compositions of NeuAc10-14Hexn+3HexNAcnFuc3 (n=16-26) with or without some O-acetylations (+42 Da) and attachment of NeuGc for NeuAc or oxidation (+16 Da). Analysis of de-N-glycosylated EPO showed the distributions of O-glycans of NeuAc1-2Hex1HexNAc1 and site occupancy. Each EPO product showed a characteristic glycoform profile with respect to sialylation, glycan size, O-acetylation of sialic acids and O-glycosylation. Analysis of darbepoetin suggested that glycans of darbepoetin were highly sialylated and O-acetylated. LC/ESI-MS was shown to be useful to evaluate the similarity of the glycoform profiles of EPO.

DOI： 10.1016/j.jpba.2013.04.031

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Vitamin B6 (pyridoxal-5'-phosphate, PLP) is linked to a variety of biological functions in prokaryotes. Here, we report that the pdxA (putative 4-hydroxy-L-threonine phosphate dehydrogenase) gene plays a pivotal role in the PLP-dependent regulation of flagellar motility, thereby altering host colonization in a leading foodborne pathogen, Campylobacter jejuni. A C. jejuni pdxA mutant failed to produce PLP and exhibited a coincident loss of flagellar motility. Mass spectrometric analyses showed a 3-fold reduction in the main flagellar glycan pseudaminic acid (Pse) associated with the disruption of pdxA. The pdxA mutant also exhibited reduced growth rates compared with the WT strain. Comparative metabolomic analyses revealed differences in respiratory/energy metabolism between WT C. jejuni and the pdxA mutant, providing a possible explanation for the differential growth fitness between the two strains. Consistent with the lack of flagellar motility, the pdxA mutant showed impaired motility-mediated responses (bacterial adhesion, ERK1/2 activation, and IL-8 production) in INT407 cells and reduced colonization of chickens compared with the WT strain. Overall, this study demonstrated that the pdxA gene affects the PLP-mediated flagellar motility function, mainly through alteration of Pse modification, and the disruption of this gene also alters the respiratory/energy metabolisms to potentially affect host colonization. Our data therefore present novel implications regarding the utility of PLP and its dependent enzymes as potent target(s) for the control of this pathogen in the poultry host. © 2013 Asakura et al.

DOI： 10.1371/journal.pone.0070418

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Language：Japanese   Publisher：日本バイオイメージング学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Human insulin and insulin lispro (lispro), a rapid-acting insulin analog, have identical primary structures, except for the transposition of a pair of amino adds. This mutation results in alterations in their higher order structures, with lispro dissociating more easily than human insulin. In our previous study performed using hydrogen/deuterium exchange mass spectrometry (HDX/MS), differences were observed in the rates and levels of deuteration among insulin analog products, which were found to be related to their self-association stability. In this study, we carried out peptide mapping of deuterated human insulin and lispro to determine the regions responsible for these deuteration differences and to elucidate the type of structural changes that affect their HDX reactivity. We identified A3-6 and B22-24 as the 2 regions that showed distinct differences in the number of deuterium atoms incorporated between human insulin and lispro. These regions contain residues that are thought to participate in hexamerization and dimerization, respectively. We also determined that over time, the differences in deuteration levels decreased in A3-6, whereas they increased in B22-24, suggesting a difference in the dynamics between these 2 regions. This article is part of a Special Issue entitled: Mass spectrometry in structural biology. (C) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbapap.2012.11.012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

We have generated a monoclonal antibody (R-10G) specific to human induced pluripotent stem (hiPS)/embryonic stem (hES) cells by using hiPS cells (Tic) as an antigen, followed by differential screening of mouse hybridomas with hiPS and human embryonal carcinoma (hEC) cells. Upon western blotting with R-10G, hiPS/ES cell lysates gave a single but an unusually diffuse band at a position corresponding to > 250 kDa. The antigen protein was isolated from the induced pluripotent stem (iPS) cell lysates with an affinity column of R-10G. The R-10G positive band was resistant to digestion with peptide N-glycanase F (PNGase F), neuraminidase, fucosidase, chondrotinase ABC and heparinase mix, but it disappeared almost completely on digestion with keratanase, keratanase II and endo-beta-galactosidase, indicating that the R-10G epitope is a keratan sulfate. The carrier protein of the R-10G epitope was identified as podocalyxin by liquid chromatography/mass spectrometry (LC/MS/MS) analysis of the R-10G positive-protein band material obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The R-10G epitope is a type of keratan sulfate with some unique properties. (1) The epitope is expressed only on hiPS/ES cells, i.e. not on hEC cells, unlike those recognized by the conventional hiPS/ES marker antibodies. (2) The epitope is a type of keratan sulfate lacking oversulfated structures and is not immunologically cross-reactive with high-sulfated keratan sulfate. (3) The R-10G epitope is distributed heterogeneously on hiPS cells, suggesting that a single colony of undifferentiated hiPS cells consists of different cell subtypes. Thus, R-10G is a novel antibody recognizing hiPS/ES cells, and should be a new molecular probe for disclosing the roles of glycans on these cells.

DOI： 10.1093/glycob/cws159

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese   Publisher：(株)じほう  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Fc gamma receptor IIa (Fc gamma RIIa) plays an important role in antibody-dependent cellular cytotoxicity and inflammation. Changes in Fc gamma RIIa expression levels or activity caused by genetic polymorphisms in FCGR2A, the gene encoding Fc gamma RIIa, may lead to differences in disease progression as well as efficacy of antibody therapeutics between individuals. In this study, we sequenced the 5'-flanking region along with all exons and their flanking regions of FCGR2A from 111 Japanese subjects. Forty-eight genetic variations were found including 12 novel ones. Beside the well-known functional 497A > G (H166R) polymorphism, we detected 818T > C (L273P) at 0.005 frequency. Since the functional significance of this polymorphism has not been revealed, we next assessed the functions of the L273P substitution by expressing wild-type and the variant proteins in human Jurkat cells. The L273P variant protein showed similar cell surface expression and IgG-binding properties as the wild-type, but it exhibited a stronger signal transduction activity based on the approximately 2-fold enhancement of tyrosine phosphorylation of Fc gamma RIIa itself. The current results suggest that L273P could have functional significance in the antibody-dependent clinical responses through Fc gamma RIIa.

DOI： 10.1007/s00251-012-0646-9

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Fcγ receptor IIa (FcγRIIa) plays an important role in antibody-dependent cellular cytotoxicity and inflammation. Changes in FcγRIIa expression levels or activity caused by genetic polymorphisms in FCGR2A, the gene encoding FcγRIIa, may lead to differences in disease progression as well as efficacy of antibody therapeutics between individuals. In this study, we sequenced the 5'-flanking region along with all exons and their flanking regions of FCGR2A from 111 Japanese subjects. Forty-eight genetic variations were found including 12 novel ones. Beside the well-known functional 497A > G (H166R) polymorphism, we detected 818T > C (L273P) at 0.005 frequency. Since the functional significance of this polymorphism has not been revealed, we next assessed the functions of the L273P substitution by expressing wild-type and the variant proteins in human Jurkat cells. The L273P variant protein showed similar cell surface expression and IgG-binding properties as the wild-type, but it exhibited a stronger signal transduction activity based on the approximately 2-fold enhancement of tyrosine phosphorylation of FcγRIIa itself. The current results suggest that L273P could have functional significance in the antibody-dependent clinical responses through FcγRIIa.

DOI： 10.1007/s00251-012-0646-9

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Language：Japanese   Publisher：(一財)医薬品医療機器レギュラトリーサイエンス財団  

ローリー法及び吸光度測定法を用いたヘパリンナトリウム及びヘパリンカルシウムのタンパク質及び核酸限度試験法について検討した。フォリン試液を2倍に希釈したときに反応溶液のpHが至適範囲に入ること、750nmにおける吸光度が最も強くなることを確認した。ヘパリンナトリウムを用いて試験法の回収率及び再現性を確認したところ良好であった。ヘパリンカルシウムは、ヘパリンナトリウムと同様に試験を実施すると反応溶液が白濁し、吸光度を測定する直前に遠心分離操作を行うこととした。この改良した試験法について適用可能性を確認した結果、回収率及び再現性ともに良好な結果が得られた。ヘパリンナトリウム及びヘパリンカルシウムの核酸限度試験としての吸光度測定法の適用可能性を検討し、ヘパリンナトリウム及びヘパリンカルシウムに含まれる0.5μg/mL程度のDNAを検出できた。

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Background: CD10, also known as neprilysin or enkephalinase exhibiting neutral endopeptidase (NEP) activity, is expressed by B-lineage hematopoietic cells as well as a variety of cells from normal tissues. It cleaves peptides such as cytokines to act for terminating inflammatory responses. Although CD10 molecules of the human pre-B-cell line NALM-6 have 6 consensus N-glycosylation sites, three of them are known to be N-glycosylated by X-ray crystallography. Methods: In order to investigate the role of N-glycans in the full expression of NEP activity, we modified N-glycans by treatment of NALM6 cells with various glycosidases or alter each of the consensus N-glycosylation sites by generating site-directed mutagenesis and compared the NEP activities of the sugar-altered CD10 with those of intact CD10. Results: CD10 of the human B-cell line NALM-6 was dominantly localized in raft microdomains and heterogeneously N-glycosylated. Although neither desialylation nor further degalactosylation caused defective NEP activity, removal of only a small part of N-glycans by treatment with glycopeptidase F under non-denaturing conditions decreased NEP activity completely. All of the three consensus sites of CD10 in HEK293 cells introduced with wild type-CD10 were confirmed to be N-glycosylated. Surface expression of N-glycan at Asn628-deleted CD10 by HEK293 cells was greatly decreased as well as it lost entire NEP activities. Conclusions: N-glycosylation at Asn628 is essential not only for NEP activities, but also for surface expression. General significance: Quality control system does not allow dysfunctional ecto-type proteases to express on plasma membrane. © 2012 Elsevier B.V.

DOI： 10.1016/j.bbagen.2012.06.017

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Language：English   Publisher：OXFORD UNIV PRESS INC  

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Language：Japanese   Publisher：(株)じほう  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

The leukocyte cell-surface antigen CD38 is the major nicotinamide adenide dinucleotide glycohydrolase in mammals, and its ectoenzyme activity is involved in calcium mobilization. CD38 is also a raft-dependent signaling molecule. CD38 forms a tetramer on the cell surface, but the structural basis and the functional significance of tetramerization have remained unexplored. We identified the interfaces contributing to the homophilic interaction of mouse CD38 by site-specific crosslinking on the cell surface with an expanded genetic code, based on a crystallographic analysis. A combination of the three interfaces enables CD38 to tetramerize: one interface involving the juxtamembrane alpha-helix is responsible for the formation of the core dimer, which is further dimerized via the other two interfaces. This dimerization of dimers is required for the catalytic activity and the localization of CD38 in membrane rafts. The glycosylation prevents further self-association of the tetramer. Accordingly, the tetrameric interaction underlies the multifaceted actions of CD38.

DOI： 10.1016/j.str.2012.06.017

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Language：Japanese   Publisher：(株)じほう  

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Language：Japanese   Publisher：(株)近代出版  

抗体医薬品の市場規模は拡大を続け、世界の医薬品市場の上位を抗体医薬品が占めるようになってきた。2000年前後に日米欧で承認された抗体医薬品製品の多くが特許期間満了となる時期を迎え、世界中で抗体医薬品のバイオ後続品/biosimilars開発への関心が高まっている。また、薬価の高い抗体医薬品の普及は、患者の経済的負担を増大させていることから、7割の薬価で供給されるバイオ後続品の普及は、患者の経済的負担を軽減させる効果があると期待されている。本稿では、我が国におけるバイオ後続品に対する規制要件と承認状況、並びに抗体医薬品バイオ後続品の展望を概説する。(著者抄録)

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Language：Japanese   Publisher：(一社)日本病院薬剤師会  

1990年前後に開発された初期のバイオ医薬品の特許期間・再審査期間が終了し、低コストでの開発が期待できる「バイオ後続品/バイオシミラー」に世界的な注目が集まるようになった。バイオ後続品の、1)新有効成分含有医薬品との違い、2)ジェネリック医薬品との違い、3)バイオシミラーとの違い、4)一般的名称、5)薬価、6)ソマトロピン後続品、7)エポエチンアルファ後続品、8)フィルグラスチム後続品、について概説した。

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jpba.2012.04.005

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Language：Japanese   Publisher：(株)じほう  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Heterozygous mutations in the JAG1 gene, encoding Notch ligand Jagged1, cause Alagille syndrome (ALGS). As most of the mutations are nonsense or frameshift mutations producing inactive truncated proteins, haplo-insufficiency is considered the major pathogenic mechanism of ALGS. However, the molecular mechanisms by which the missense mutations cause ALGS remain unclear. Here we analyzed the functional properties of four ALGS missense mutant proteins, P163L, R184H, G386R and C714Y, using transfected mammalian cells. P163L and R184H showed Notch-binding activities similar to that of the wild-type when assessed by immunoprecipitation. However, their trans-activation and cis-inhibition activities were almost completely impaired. These mutant proteins localized mainly to the endoplasmic reticulum (ER), suggesting that the mutations induced improper protein folding. Furthermore, the mutant proteins bound more strongly to the ER chaperone proteins calnexin and calreticulin than the wild-type did. C714Y also localized to the ER, but possessed significant trans-activation activity and lacked enhanced binding to the chaperones, indicating a less severe phenotype. The properties of G386R were the same as those of the wild-type. Dominant-negative effects were not detected for any mutant protein. These results indicate that accumulation in the ER and binding to the chaperones correlate with the impaired signal-transduction activities of the missense mutant proteins, which may contribute to the pathogenic mechanism of ALGS. Our findings, which suggest the requirement for cell-surface localization of Jagged1 for cis-inhibition activities, also provide important information for understanding the molecular basis of Notch-signaling pathways.

DOI： 10.1111/j.1742-4658.2012.08595.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Porcine pancreatic alpha-amylase (PPA) binds to N-linked glycans of glycoproteins (Matsushita, H., Takenaka, M., and Ogawa, H. (2002) J. Biol Chem., 277, 4680-4686). Immunostaining revealed that PPA is located at the brush-border membrane (BBM) of enterocytes in the duodenum and that the binding is inhibited by mannan but not galactan, indicating that PPA binds carbohydrate-specifically to BBM. The ligands for PPA in BBM were identified as glycoprotein N-glycans that are significantly involved in the assimilation of glucose, including sucrase-isomaltase (SI) and Na+/Glc cotransporter 1 (SGLT1). Binding of SI and SGLT1 in BBM to PPA was dose-dependent and inhibited by mannan. Using BBM vesicles, we found functional changes in PPA and its ligands in BBM due to the N-glycanspecific interaction. The starch-degrading activity of PPA and maltose-degrading activity of SI were enhanced to 240 and 175%, respectively, while Glc uptake by SGLT1 was markedly inhibited by PPA at high but physiologically possible concentrations, and the binding was attenuated by the addition of mannose-specific lectins, especially from Galanthus nivalis. Additionally, recombinant human pancreatic alpha-amylases expressed in yeast and purified by single-step affinity chromatography exhibited the same carbohydrate binding specificity as PPA in binding assays with sugar-biotinyl polymer probes. The results indicate that mammalian pancreatic alpha-amylases share a common carbohydrate binding activity and specifically bind to the intestinal BBM. Interaction with N-glycans in the BBM activated PPA and SI to produce much Glc on the one hand and to inhibit Glc absorption by enterocytes via SGLT1 in order to prevent a rapid increase in blood sugar on the other.

DOI： 10.1074/jbc.M111.314658

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Aberrant glycosylation has been observed in many types of cancer, but the mechanism of glycosylation change is still poorly understood. To elucidate relationships between glycosylation and colon cancer progression, we analyzed glycosylation status of beta-haptoglobin (beta-Hp) obtained from 46 cancer patients, 14 inflammatory bowel disease patients and 38 normal subjects. Aleuria aurantia lectin reactivity with cancer beta-Hp was much higher than in the other two study groups. These results were confirmed by lectin blotting and microarray assay using other lectins directed to fucosyl residues. Levels of such glycans were correlated with stage of colon cancer progression. Reactivity with fucosylated glycans was eliminated by treatment with a1-3/4 fucosidase but not a1-6 fucosidase, indicating that enhanced lectin reactivity with the fucose moiety of colon cancer beta-Hp is due to Fuca1-3/4GlcNAc. Moreover, site-specific glycan occupancy was determined by sequential LC/MS analysis. Mass spectrometric analysis showed that fucosylation of beta-Hp was higher in colon cancer patients than in other subjects. In particular, fucosylation at Asn 241 of beta-Hp in sera of colon cancer patients was clearly higher than in the other groups, and the ratio of fucosylated glycopeptides containing Asn 241 decreased greatly after treatment with a1-3/4 fucosidase. In conclusion, the level of a1-3/4 fucosyl epitope at Asn 241 of beta-Hp is potentially useful as a novel marker for colon cancer.

DOI： 10.1002/ijc.26288

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Language：Japanese   Publisher：(株)じほう  

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A conjugate of polyl-lysine (PLL) with unsulfated dextran produced by reductive amination was found to have remarkable anti-HIV-1 activity against both the macrophage-tropic R5 virus Ba-L and T-cell line tropic X4 virus IIIB strains, although neither PLL nor dextran has such activity. The conjugate is a pseudoproteoglycan (pseudoPG) that simulates the structure of a proteoglycan. Conjugation with dextran was found to produce an antiviral effect in three kinds of assay systems including a human CD4 + T-cell line, and the pseudoPG synthesized using 10kDa PLL and 10kDa dextran showed EC 50 4-40 times lower than that of sulfated dextran or heparin against Ba-L and EC 50 equal to that against IIIB, indicating that PLL-dextran (PLL-Dex) was more effective against R5 virus than sulfated polysaccharides. PLL-Dex significantly suppressed a clinically isolated R5 virus from primary peripheral blood mononuclear cells. PLL-Dex interacted with the virus during adsorption to the cell and also decreased virus entry into the cell, suggesting PLL-Dex has multiple preventive mechanisms against HIV-1. © 2012 Elsevier B.V.

DOI： 10.1016/j.antiviral.2012.02.011

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Glycosylphosphatidylinositol (GPI)-anchor attachment is one of the most common posttranslational protein modifications. Using the nematode Caenorhabditis elegans, we determined that GPI-anchored proteins are present in germline cells and distal tip cells, which are essential for the maintenance of the germline stem cell niche. We identified 24 C. elegans genes involved in GPI-anchor synthesis. Inhibition of various steps of GPI-anchor synthesis by RNA interference or gene knockout resulted in abnormal development of oocytes and early embryos, and both lethal and sterile phenotypes were observed. The piga-1 gene (orthologue of human PIGA) codes for the catalytic subunit of the phosphatidylinositol N-acetylglucosaminyltransferase complex, which catalyzes the first step of GPI-anchor synthesis. We isolated piga-1 - knockout worms and found that GPI-anchor synthesis is indispensable for the maintenance of mitotic germline cell number. The knockout worms displayed 100% lethality, with decreased mitotic germline cells and abnormal eggshell formation. Using cell-specific rescue of the null allele, we showed that expression of piga-1 in somatic gonads and/or in germline is sufficient for normal embryonic development and the maintenance of the germline mitotic cells. These results clearly demonstrate that GPI-anchor synthesis is indispensable for germline formation and for normal development of oocytes and eggs. © 2012 Murata et al.

DOI： 10.1091/mbc.E10-10-0855

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：1  

Insulin analog products for subcutaneous injection are prepared as solutions in which insulin analog molecules exist in several oligomeric states. Oligomeric stability can affect their onset and duration of action and has been exploited in designing them. To investigate the oligomeric stability of insulin analog products having different pharmacokinetics, we performed hydrogen/deuterium exchange mass spectrometry (HDX/MS), which is a rapid method to analyze dynamic aspects of protein structures. Two rapid-acting analogs (lispro and glulisine) incorporated deuteriums more and faster than recombinant human insulin, whereas a long-acting analog (glargine) and two intermediate-acting preparations (protamine-containing formulations) incorporated them less and more slowly. Kinetic analysis revealed that the number of slowly exchanged hydrogens (D(s)) (k<0.01 min(-1)) accounted for the difference in HDX reactivity among analogs. Furthermore, we found correlations between HDX kinetics and pharmacokinetics reported previously. Their maximum serum concentration (C(max)) was linearly correlated with D(s) (r=0.88) and the number of maximum exchangeable hydrogens (D(∞)) (r=0.89). The maximum drug concentration time (t(max)) was also correlated with reciprocals of D(s) and D(∞) (r=0.86 and r=0.96, respectively). Here we demonstrate the ability of HDX/MS to evaluate oligomeric stability of insulin analog products.

DOI： 10.1016/j.ab.2011.09.002

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Language：Japanese   Publisher：(一社)レギュラトリーサイエンス学会  

バイオ医薬品は、肝炎、糖尿病、及びある種の血液関連疾患やがん領域において標準的治療薬となっており、その医療上の重要性は増す一方である。治療法のない疾患への新たな治療法の提供やQOLの向上を目的として、新規な医薬品開発への期待はさらに高まっている。規制側からも、ICH Q11案、ICHS6(R1)案、並びに「治験対象医薬品ヒト初回投与試験の安全性に関するガイダンス(FIH)案」などが立て続けに示された。Q11案では、重要品質特性を特定するために、徹底した特性解析が重要であること、また、S6(R1)案及びFIH案においても、新規な分子構造、複合型機能、及び高い分子標的性を有するバイオ医薬品及びその被験薬の安全性確保には、特性への十分な理解が不可欠であることが示された。新規なバイオ医薬品の研究開発から生産に至る過程において、作用機構と安全性の予測につながる新たな特性解析法の開発の必要性が高まっている。(著者抄録)

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Language：Japanese   Publisher：4  

Glycosylation of cells is known to alter with several biological events such as cell differentiations and proliferations as well as some diseases. "Glycomic approaches", comprehensive qualitative and quantitative glycan analyses of the cells, have become increasingly important as a means of discovering biomarkers that have the potential of being used as disease diagnostic markers and molecular markers for cell characterizations. In this paper, we introduce a method of quantitative glycan profiling by liquid chromatography/mass spectrometry with a combination of an isotope tagging method. In addition, we demonstrate the potential of glycan profiling as a tool for the identification of differentiated human bone marrow mesenchymal stem cell (hMSC) and non-differentiated hMSC.<br>

DOI： 10.1248/yakushi.132.489

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Other Link： https://jlc.jst.go.jp/DN/JALC/10000140880?from=CiNii

Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We previously reported that the ascidian sperm proteasome degrades the egg-coat protein extracellularly during fertilization. In order to explore an extracellular transport signal, we purified the proteasome from ascidian sperm and compared its subunit structure with egg and muscle proteasomes. The results showed that PSMA1/alpha 6 subunit of the sperm proteasome is distinct from egg and muscle proteasomes. LC/MS/MS analysis revealed that the C-terminal 16 residues of sperm alpha 6 subunit are processed. Whereas sperm-specific paralogous genes of alpha subunits are reported, its sperm-specific C-terminal processing is a newly discovered novel post-translational modification of the proteasome. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.06.069

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Dendritic cell (DC)-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is a type II transmembrane C-type lectin expressed on DCs such as myeloid DCs and monocyte-derived DCs (MoDCs). Recently, we have reported that DC-SIGN interacts with carcinoembryonic antigen (CEA) expressed on colorectal carcinoma cells. CEA is one of the most widely used tumor markers for gastrointestinal cancers such as colorectal cancer. On the other hand, other groups have reported that the level of Mac-2-binding protein (Mac-2BP) increases in patients with pancreatic, breast, and lung cancers, virus infections such as human immunodeficiency virus and hepatitis C virus, and autoimmune diseases. Here, we first identified Mac-2BP expressed on several colorectal carcinoma cell lines as a novel DC-SIGN ligand through affinity chromatography and mass spectrometry. Interestingly, we found that DC-SIGN selectively recognizes Mac-2BP derived from some colorectal carcinomas but not from the other ones. Furthermore, we found that the alpha 1-3,4-fucose moieties of Le glycans expressed on DC-SIGN-binding Mac-2BP were important for recognition. DC-SIGN-dependent cellular interactions between immature MoDCs and colorectal carcinoma cells significantly inhibited MoDC functional maturation, suggesting that Mac-2BP may provide a tolerogenic microenvironment for colorectal carcinoma cells through DC-SIGN-dependent recognition. Importantly, Mac-2BP was detected as a predominant DC-SIGN

DOI： 10.1074/jbc.M110.215301

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4-7 N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAECPAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix.
Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study. (C) 2011 The International Alliance for Biologicals. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biologicals.2011.04.002

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Language：Japanese   Publisher：(一財)医薬品医療機器レギュラトリーサイエンス財団  

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Helicobacter pylori is a human gastric pathogen associated with gastric and duodenal ulcers as well as gastric cancer. Mounting evidence suggests this pathogen's motility is prerequisite for successful colonization of human gastric tissues. Here, we isolated an H. pylori G27 HP0518 mutant exhibiting altered motility in comparison to its parental strain. We show that the mutant's modulated motility is linked to increased levels of O-linked glycosylation on flagellin A (FlaA) protein. Recombinant HP0518 protein decreased glycosylation levels of H. pylori flagellin in vitro, indicating that HP0518 functions in deglycosylation of FlaA protein. Furthermore, mass spectrometric analysis revealed increased glycosylation of HP0518 FlaA was due to a change in pseudaminic acid (Pse) levels on FlaA; HP0518 mutant-derived flagellin contained approximately threefold more Pse than the parental strain. Further phenotypic and molecular characterization demonstrated that the hyper-motile HP0518 mutant exhibits superior colonization capabilities and subsequently triggers enhanced CagA phosphorylation and NF-κB activation in AGS cells. Our study shows that HP0518 is involved in the deglycosylation of flagellin, thereby regulating pathogen motility. These findings corroborate the prominent function of H. pylori flagella in pathogen-host cell interactions and modulation of host cell responses, likely influencing the pathogenesis process.

DOI： 10.1111/j.1365-2958.2010.07393.x

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Language：Japanese   Publisher：(株)じほう  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Heparin sodium and heparin calcium, which are widely used as anti-coagulants, are known to potentially contain the natural impurity dermatan sulfate (DS). Recently serious adverse events occurred in patients receiving heparin sodium in the US, and a contaminant oversulfated chondroitin sulfate (OSCS) was found to be a cause of the events. To ensure the quality and safety of pharmaceutical heparins, there is need of a physicochemical identification test that can discriminate heparin from the heparin-related substances as well as a sensitive purity test for OSCS. Recently, HPLC with a strong-anion exchange column was proposed as the methods for identifying heparin and determination of OSCS in heparin sodium. Although this method is convenient and easy to perform, the only column suitable for this purpose is the Dionex IonPac AS11-HC column. In this study, we developed alternative identification test and test for OSCS in both heparin sodium and heparin calcium using a weak anion-exchange column. The identification test allowed for separation of heparin from the impurity DS and contaminant OSCS in a shorter time. The purity test provided enough sensitivity, specificity, linearity, recovery and repeatability for OSCS. We believe that our methods will be useful for quality control of pharmaceutical heparins. (C) 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biologicals.2010.04.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The extracellular matrix (ECM) molecules play important roles in many biological and pathological processes. During tissue remodeling, the ECM molecules that are glycosylated are different from those of normal tissue owing to changes in the expression of many proteins that are responsible for glycan synthesis. Vitronectin (VN) is a major ECM molecule that recognizes integrin on hepatic stellate cells (HSCs). The present study attempted to elucidate how changes in VN glycans modulate the survival of HSCs, which play a critical role in liver regeneration. Plasma VN was purified from partially hepatectomized (PH) and sham-operated (SH) rats at 24 h after operation and non-operated (NO) rats. Adhesion of rat HSCs (rHSCs), together with phosphorylation of focal adhesion kinase, in PH-VN was decreased to one-half of that in NO-or SH-VN. Spreading of rHSCs on desialylated NO-VN was decreased to one-half of that of control VN, indicating the importance of sialylation of VN for activation of HSCs. Liquid chromatography/multiple-stage mass spectrometry analysis of Glu-C glycopeptides of each VN determined the site-specific glycosylation. In addition to the major biantennary complex-type N-glycans, hybrid-type N-glycans were site-specifically present at Asn(167). Highly sialylated O-glycans were found to be present in the Thr(110)-Thr(124) region. In PH-VN, the disialyl O-glycans and complex-type N-glycans were decreased while core-fucosylated N-glycans were increased. In addition, immunodetection after two-dimensional PAGE indicated the presence of hyper- and hyposialylated molecules in each VN and showed that hypersialylation was markedly attenuated in PH-VN. This study proposes that the alteration of VN glycosylation modulates the substrate adhesion to rat HSCs, which is responsible for matrix restructuring.

DOI： 10.1074/jbc.M109.077016

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The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

DOI： 10.1074/mcp.M900450-MCP200

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Complete NMR analysis of oxytocin (OXT) in phosphate buffer was elucidated by one-dimensional (1D)-and two-dimensional (2D)-NMR techniques, which involve the assignment of peptide amide NH protons and carbamoyl NH2 protons. The1H-15N correlation of seven amide NH protons and three carbamoyl NH2 protons were also shown by HSQC NMR of OXT without15N enrichment. Copyright © 2009 John Wiley &amp
Sons, Ltd.

DOI： 10.1002/mrc.2557

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Neonatal Fc receptor (FcRn) plays an important role in regulating IgG homeostasis in the body. Changes in FcRn expression levels or activity caused by genetic polymorphisms of FCGRT, which encodes FcRn, may lead to interindividual differences in pharmacokinetics of therapeutic antibodies. In this study, we sequenced the 5'-flanking region, all exons and their flanking regions of FCGRT from 126 Japanese subjects. Thirty-three genetic variations, including 17 novel ones, were found. Of these, two novel non-synonymous variations, 629G>A (R210Q) and 889T>A (S297T), were found as heterozygous variations. We next assessed the functional significance of the two novel non-synonymous variations by expressing wild-type and variant proteins in HeLa cells. Both variant proteins showed similar intracellular localization as well as antibody recycling efficiencies. These results suggested that at least no common functional polymorphic site with amino acid change was present in the FCGRT of our Japanese population.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC STUDY XENOBIOTICS  

Neonatal Fc receptor (FcRn) plays an important role in regulating IgG homeostasis in the body. Changes in FcRn expression levels or activity caused by genetic polymorphisms of FCGRT, which encodes FcRn, may lead to interindividual differences in pharmacokinetics of therapeutic antibodies. In this study, we sequenced the 5'-flanking region, all exons and their flanking regions of FCGRT from 126 Japanese subjects. Thirty-three genetic variations, including 17 novel ones, were found. Of these, two novel non-synonymous variations, 629G > A (R210Q) and 889T > A (S297T), were found as heterozygous variations. We next assessed the functional significance of the two novel non-synonymous variations by expressing wild-type and variant proteins in HeLa cells. Both variant proteins showed similar intracellular localization as well as antibody recycling efficiencies. These results suggested that at least no common functional polymorphic site with amino acid change was present in the FCGRT of our Japanese population.

DOI： 10.2133/dmpk.DMPK-10-RG-067

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A new method that combines 1H-NMR and principal component analysis (PCA) was employed to obtain the quality evaluation of biopharmaceuticals, with regard to their quality, consistency, and differences in protein modification patterns. To assess the feasibility of the method, three 1H-NMR spectra of oxytocin (OXT) were collected every 7 d (at Day 0, 7 and 14), and time-dependent changes in the spectra were found by PCA of the 1H-NMR signals from 0.5-9.0 ppm, excluding the region around the water signal (4.6-5.0 ppm). Although the three OXT spectra seemed similar by simple visual inspection, time-dependent differences among the three spectra were clearly distinguished by a PCA scores plot. Peak changes indicating both OXT decomposition and the emergence of new OXT decomposition products within the timeframe of the experiment were also observed by a PCA loading plot. The results demonstrate that this method can evaluate the consistency of biopharmaceutical quality. © 2009 Pharmaceutical Society of Japan.

DOI： 10.1248/cpb.57.1396

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Genetically modified (GM) foods are beneficial from the standpoint of ensuring a constant supply of foodstuffs, but they must be tested for safety before being released on the market, including by allergenicity tests to ensure that they do not contain new allergens or higher concentrations of known allergens than the same non-GM foods. In this study we used GM-amago salmon into which a growth hormone gene had been introduced and compared the allergens contained in the GM and the non-GM-amago salmons. We used a combination of Western blotting with allergen-specific antibodies and a proteomic analysis of their allergens with patients' sera, a so-called allergenome analysis, to analyze allergens. Western blotting with specific antibodies showed no increase in the content of the known allergens fish parvalbumin and fish type-I collagen in GM-amago salmon, in comparison with their content in non-GM-amago salmon. The allergenome analysis of two fish-allergic patients allowed us to identify several IgE-binding proteins in amago salmon, including parvalbumin, triose-phosphate isomerase, fructose-bisphosphate aldolase A, and serum albumin, and there were no qualitative differences in these proteins between GM and non-GM-amago salmons. These results indicate that amago salmon endogenous allergen expression does not seem to be altered by genetic modification. © 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yrtph.2009.08.002

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HNK-1 (human natural killer-1) glyco-epitope, a sulfated glucuronic acid attached to N-acetyllactosamine on the nonreducing termini of glycans, is highly expressed in the nervous system. Our previous report showed that mice lacking a glucuronyltransferase (GlcAT-P), a key enzyme for biosynthesis of the HNK-1 epitope, showed reduced long term potentiation at hippocampal CA1 synapses. In this study, we identified an α-amino-3-hydroxy-5-methylisoxazole propionate (AMPA)-type glutamate receptor subunit, GluR2, which directly contributes to excitatory synaptic transmission and synaptic plasticity, as a novel HNK-1 carrier molecule. We demonstrated that the HNK-1 epitope is specifically expressed on the N-linked glycan(s) on GluR2 among the glutamate receptors tested, and the glycan structure, including HNK-1 on GluR2, was determined using liquid chromatography-tandem mass spectrometry. As for the function of HNK-1 on GluR2, we found that the GluR2 not carrying HNK-1 was dramatically endocytosed and expressed less on the cell surface compared with GluR2 carrying HNK-1 in both cultured hippocampal neurons and heterologous cells. These results suggest that HNK-1 stabilizes GluR2 on neuronal surface membranes and regulates the number of surface AMP A receptors. Moreover, we showed that the expression of the HNK-1 epitope enhanced the interaction between GluR2 and N-cadherin, which has important roles in AMPA receptor trafficking. Our findings suggest that the HNK-1 epitope on GluR2 regulates cell surface stability of GluR2 by modulating the interaction with N-cadherin. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M109.024208

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：PHARMACEUTICAL SOC JAPAN  

Heparin is widely used as an anticoagulant for the treatment and prevention of thrombotic disorders. Recently, hundreds of cases of anaphylactic reaction as adverse effects were reported by the presence of contaminating oversulfated chondroitin sulfate (OSCS) in some heparin preparations. In addition, these heparin preparations often contaminated dermatan sulfate (DS). Unfortunately, the Japanese Pharmacopoeia (JP) does not include appropriate purity tests. In the present paper, we show that capillary electrophoresis (CE) is a powerful tool for the analysis of OSCS and DS ill heparin preparations. CE method shows high resolution and good quantification of OSCS in heparin preparations. This method (OSCS method) was evaluated for accuracy (93.7%), repeatability (R.S.D. = 2.11), linearity (R-2 = 0.9996), detection limit (0.1% OSCS) and specificity. In contrast, DS was not able to be detected in high sensitivity by OSCS method. However, a modified CE method (DS method) using the buffer at lower pHs showed good parameters for accuracy (88.1%,), repeatability (R.S.D. = 1.99), linearity (R-2 = 0.9998), detection limit (0.25% DS) and specificity. In conclusion, CE will be an alternative to the NMR method which is being adopted for purification test of heparin sodium in the present version of JP.

DOI： 10.1248/yakushi.129.1255

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：7  

Certain glycan motifs in glycoproteins are involved in several biological events and diseases. To understand the roles of these motifs, a method is needed to identify the glycoproteins that carry them. We previously demonstrated that liquid chromatography-multiple-stage mass spectrometry (LC-MSn) allowed for differentiation of oligosaccharides attached to Lewis-motifs, such as Lewisx(Lex, Galbeta1-4(Fucalpha1-3)GlcNAc) from other glycans. We successfully discriminated Lex-conjugated oligosaccharides from other N-linked oligosaccharides derived from mouse kidney proteins by using Lewis-motif-distinctive ions, a deoxyhexose (dHex)+hexose (Hex)+N-acetylhexsosamine (HexNAc) fragment (m/z 512), and a Hex+HexNAc fragment (m/z 366). In the present study, we demonstrated that this method could be used to identify the Lex-conjugated glycoproteins. All proteins in the mouse kidney were digested into peptides, and the fucosylated glycopeptides were enriched by lectin-affinity chromatography. The resulting fucosylated glycopeptides were subjected to two different runs of LC-MSn using a Fourier- transform ion cyclotron resonance mass spectrometer (FTICR-MS) and an ion trap-type mass spectrometer. After the first run, we picked out product ion spectra of the expected Lex-conjugated glycopeptides based on the presence of Lewis-motif-distinctive ions and assigned a peptide+HexNAc or peptide+(dHex)HexNAc fragment in each spectrum. Then the fucosylated glycopeptides were subjected to a second run in which the peptide-related fragments were set as precursor ions. We successfully identified gamma-glutamyl transpeptidase 1 (gamma-GTP1), low-density lipoprotein receptor-related protein 2 (LRP2), and a cubilin precursor as Lex-conjugated glycoproteins by sequencing of 2-5 glycopeptides. In addition, it was deduced that cadherin 16, dipeptidase I, H-2 class I histocompatibility antigen, K-K alpha precursor (H2-Kk), and alanyl (membrane) aminopeptidase could be Lex-conjugated glycoproteins from the good agreement between the experimental and theoretical masses and fragment patterns. The results indicated that our method could be applicable for the identification and screening of glycoproteins carrying target glycan-motifs, such as Lewis epitopes.

DOI： 10.1021/pr9000527

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHARMACEUTICAL SOC JAPAN  

Many glycoproteins and glycosaminoglycans are approved for clinical use. Carbohydrate moieties in biopharmaceuticals affect not only their physicochemical properties and thermal stability, but also their reactivity with their receptors and circulating half-life. Modification of glycans is one target of drug design for enhancement of efficacy. Meanwhile, there have been reports of serious adverse events caused by some carbohydrates. It is crucial to maintain the constancy of carbohydrate moieties for the efficient and safe use of glycosylated biopharmaceuticals. On the other hand, for scientific, safety-related, and economic reasons, changes in the manufacturing process are frequently made either during the development or after the approval of new biopharmaceuticals. Furthermore, the development of biosimilar glycoprotein products has been attempted by different manufacturers. Changes in pharmaceutical manufacturing processes possibly cause alteration of glycosylation and raise concerns about alteration of their quality, safety, and efficacy. In this review we provide some current topics of glycosylated biopharmaceuticals from the viewpoints of efficacy, safety, and the manufacturing process and discuss the significance of glycosylation analysis for development of biopharmaceuticals.

DOI： 10.1248/bpb.32.796

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Changes in the glycan structures of some glycoproteins have been observed in autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis. A deficiency of alpha-mannosidase II, which is associated with branching in N-glycans, has been found to induce SLE-like glomerular nephritis in a mouse model. These findings suggest that the alteration of the glycosylation has some link with the development of SLE. An analysis of glycan alteration in the disordered tissues in SLE may lead to the development of improved diagnostic methods and may help to clarify the carbohydrate-related pathogenic mechanism of inflammation in SLE. In this study, a comprehensive and differential analysis of N-glycans in kidneys from SLE-model mice and control mice was performed by using the quantitative glycan profiling method that we have developed previously. In this method, a mixture of deuterium-labelled N-glycans from the kidneys of SLE-model mice and non-labelled N-glycans from kidneys of control mice was analysed by liquid chromatography/mass spectrometry. It was revealed that the low-molecular-mass glycans with simple structures, including agalactobiantennary and paucimannose-type oligosaccharides, markedly increased in the SLE-model mouse. On the other hand, fucosylated and galactosylated complex type glycans with high branching were decreased in the SLE-model mouse. These results suggest that the changes occurring in the N-glycan synthesis pathway may cause the aberrant glycosylations on not only specific glycoproteins but also on most of the glycoproteins in the SLE-model mouse. The changes in glycosylation might be involved in autoimmune pathogenesis in the model mouse kidney.

DOI： 10.1111/j.1365-2567.2008.02898.x

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Liquid chromatography/multiple-stage mass spectrometry (LC/MSn) is an effective means for the site-specific glycosylation analysis of a limited quantity of glycoproteins, such as gel-separated proteins. Generally, a tryptic digest of the glycoprotein is separated by reversed-phase LC, and peptide sequencing and glycosylation analysis are achieved with on-line MSn. In this chapter, a protocol for the LC/MS/MS/MS of a proteolytic digest of a gel-separated glycoprotein is described. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.

DOI： 10.1007/978-1-59745-022-5_18

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The serum mannan-binding protein (MBP) is a host defense C-type lectin specific for mannose, N-acetylglucosamine, and fucose residues, and exhibits growth inhibitory activity toward human colorectal carcinoma cells. The MBP-ligand oligosaccharides (MLO) isolated from a human colorectal carcinoma cell line, SW1116, are large, multiantennary N-glycans with highly fucosylated polylactosamine-type structures having Leb Lea or tandem repeats of the Lea structure at their nonreducing ends. In this study, we isolated the major MBP-ligand glycoproteins from SW1116 cell lysates with an MBP column and identified them as CD26/dipeptidyl peptidase IV (DPPIV) (110 kDa) and CD98 heavy chain (CD98hc)/4F2hc (82 kDa). Glycosidase digestion revealed that CD26 contained such complex-type N-glycans that appear to mediate the MBP binding. MALDI-MS of the N-glycans released from CD26 by PNGase F demonstrated conclusively that CD26 is the major MLO-carrying protein. More interestingly, a comparison of the N-glycans released from the MBP-binding and non-MBP-binding glycopeptides suggested that complex-type N-glycans carrying a minimum of 4 Lea/ Leb epitopes arranged either as multimeric tandem repeats or terminal epitopes on multiantennary structures are critically important for the high affinity binding to MBP. Analysis of the N-glycan attachment sites demonstrated that the high affinity MLO was expressed preferentially at some N-glycosylation sites, but this site preference was not so stringent. Finally, hypothetical 3D models of tandem repeats of the Lea epitope and the MBP-Lewis oligosaccharide complex were presented. © The Author 2009. Published by Oxford University Press. All rights reserved.

DOI： 10.1093/glycob/cwn158

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Laminin-332 (Lm332) is a large heterotrimeric glycoprotein that has been identified as a scattering factor, a regulator of cancer invasion as well as a prominent basement membrane component of the skin. Past studies have identified the functional domains of Lm332 and revealed the relationships between its activities and the processing of its subunits. However, there is little information available concerning the effects of N-glycosylation on Lm332 activities. In some cancer cells, an increase of β1,6-GlcNAc catalyzed by N-acetylglucosaminyltransferase V (GnT-V) is related to the promotion of cancer cell motility. By contrast, bisecting GlcNAc catalyzed by N- acetylglucosaminyltransferase III (GnT-III) suppresses the further processing with branching enzymes, such as GnT-V, and the elongation of N-glycans. To examine the effects of those N-glycosylations to Lm332 on its activities, we purified Lm332s from the conditioned media of GnT-III- and GnT-V-overexpressing MKN45 cells. Lectin blotting and mass spectrometry analyses revealed that N-glycans containing the bisecting GlcNAc and β1,6-GlcNAc structures were strongly expressed on Lm332 purified from GnT-III-overexpressing (GnT-III-Lm332) and GnT-V-overexpressing (GnTV-Lm332) cells, respectively. Interestingly, the cell adhesion activity of GnT-III-Lm332 was apparently decreased compared with those of control Lm332 and GnT-V-Lm332. In addition, the introduction of bisecting GlcNAc to Lm332 resulted in a decrease in its cell scattering and migration activities. The weakened activities were most likely derived from the impaired α3β1 integrin clustering and resultant focal adhesion formation. Taken together, our results clearly demonstrate for the first time that N-glycosylation may regulate the biological function of Lm332. This finding could introduce a new therapeutic strategy for cancer. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M804526200

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Language：Japanese   Publisher：12 Suppl  

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Other Link： http://search.jamas.or.jp/link/ui/2008351288

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Fibronectin (FN) is a multifunctional glycoprotein present in the extracellular matrix (ECM) and plasma. We previously reported that the glycosylation and ligand-binding of vitronectin (VN) change markedly after partial hepatectomy (PH). Here we show the changes of FN during liver regeneration. The yields of purified sham-operated (SH-) and PH-FN were higher than that of non-operated (NO)-FN, while binding activities of FNs to ECM ligands were changed only slightly by hepatectomy. The carbohydrate concentration of PH-FN decreased to 66% of that of NO- and SH-FN. By using LC/MS '', eight kinds of complex-type N-glycan structures were found to be present in all FNs, and bi- and trisialobiantennary glycans were the major structures. Fucosylation was markedly increased, while O-acetylation of sialic acid was found to be decreased in PH-FN. The alterations in glycosylation and biological activities of FN after PH are different from those of VN, suggesting that these glycoproteins play different biological functions in tissue remodeling. (C) 2008 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2008.03.027

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

IgLON family proteins, including limbic-associated membrane protein (LAMP), opioid-binding cell adhesion molecule (OBCAM), neurotrimin, and Kilon, are immunoglobulin (Ig) superfamily cell adhesion molecules. These molecules are composed of three Ig domains and a glycosylphosphatidylinositol (GPI) anchor and contain six or seven potential N-glycosylation sites. Although their glycosylations are supposed to be associated with the development of the central nervous system like other Ig superfamily proteins, they are still unknown because of difficulty in isolating individual proteins with a high degree of homology in performing carbohydrate analysis. In this study, we conducted simultaneous site-specific glycosylation analysis of rat brain IgLON proteins by liquid chromatography and multiple-stage mass spectrometry (LC-MSn). The rat brain GPI-linked proteins were enriched and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The four proteins were extracted from the gel, and subjected to LC-MSn after proteinase digestions. A set of glycopeptide MS data, including the mass spectrum, the mass spectrum in the selected ion monitoring mode, and the product ion spectra, was selected from all data based on carbohydrate-related ions in the MS/MS spectrum. The peptide portion and the carbohydrate structure were identified on the basis of peptide-related ion and carbohydrate-related ions, and the accurate mass. The site-specific glycosylations of four proteins were elucidated as follows. N-Glycans near the N-terminal were disialic acid-conjugated complex- and hybrid-type oligosaccharides. The first Ig domains were occupied by Man-5-9. Diverse oligosaccharides, including Lewis a/x-modified glycans, a brainspecific glycan known as BA-2, and Man-5, were found to be attached to the third Ig domain. Three common structures of glycans were found in the GPI moiety of LAMP, OBCAM, and neurotrimin.

DOI： 10.1021/bi8009778

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Although ascidians are hermaphroditic, many species including Halocynthia roretzi are self-sterile. We previously reported that a vitelline coat polymorphic protein HrVC70, consisting of 12 EGF (epidermal growth factor)-like repeats, is a candidate allorecognition protein in H. roretzi, because the isolated HrVC70 shows higher affinity to nonself-sperm than to self-sperm. Here, we show that a sperm 35-kDa glycosylphosphatidylinositol-anchored CRISP (cysteine-rich secretory protein)-like protein HrUrabin in a low density detergent-insoluble membrane fraction is a physiological binding partner for HrVC70. We found that HrVC70 specifically interacts with HrUrabin, which had been separated by SDS-PAGE and transferred onto a nitrocellulose membrane. HrUrabin has an N-linked sugar chain, essential for binding to HrVC70. Hr-Urabin mRNA is expressed in the testis but not in the ovary, and the protein appears to be localized on the surface of sperm head and tail. Anti-HrUrabin antibody, which neutralizes the interaction between HrUrabin and HrVC70, potently inhibited fertilization and allorecognizable sperm-binding to HrVC70-agarose. However, no significant difference in the binding ability of HrUrabin to HrVC70 was observed in autologous and allogeneic combinations by Far Western analyses. These results indicate that sperm-egg binding in H. roretzi is mediated by the molecular interaction between HrUrabin on the sperm surface and HrVC70 on the vitelline coat, but that HrUrabin per se is unlikely to be a direct allorecognition protein. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M802631200

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Language：English   Publisher：1-2  

DOI： 10.1016/j.jchromb.2008.05.006

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The HNK-1 epitope has a unique structure comprising the sulfated trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc), and two glucuronyltransferases (GlcAT-P and GlcAT-S) are key enzymes for its biosynthesis. However, the different functional roles of these enzymes in its biosynthesis remain unclear. Recently, we reported that a nonsulfated form of this epitope, which is biosynthesized by GlcAT-S but not by GlcAT-P, is expressed on two metalloproteases in mouse kidney. In this study, we found that a novel glycoprotein carrying the nonsulfated HNK-1 epitope in mouse kidney was enriched in the nuclear fraction. The protein was affinity-purified and identified as laminin-1, and we also confirmed the N -linked oligosaccharide structure including nonsulfated HNK-1 epitope derived from laminin-1 by mass spectrometry. Curiously, immunofluorescence staining of kidney sections revealed that laminin-1 appeared not to be colocalized with the nonsulfated HNK-1 epitope. However, proteinase treatment strengthened the signals of both laminin-1 and the nonsulfated HNK-1 epitope, resulting in overlapping of them. These results indicate that the nonsulfated HNK-1 epitope on laminin-1 is usually embedded and masked in the robust basement membrane in tight association with other proteins. To clarify the associated proteins and the functional role of the carbohydrate epitope, we investigated the interaction between laminin-1 and alpha-dystroglycan through their glycans in mouse kidney using the overlay assay technique. We obtained evidence that glucuronic acid as well as sialic acid inhibited this interaction, suggesting that the nonsulfated HNK-1 epitope on laminin-1 may regulate its binding and play a role in maintenance of the proper structure in the kidney basal lamina. © The Author 2008. Published by Oxford University Press. All rights reserved.

DOI： 10.1093/glycob/cwn012

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N-Glycolylneuraminic acid (NeuGc), an acidic nine-carbon sugar, is produced in several animals, such as cattle and mice. Since human cells cannot synthesize NeuGc, it is considered to be immunogenic in humans. Recently, NeuGc contamination was reported in human embryonic stem cells cultured with xenogeneic serum and cells, suggesting that possibly NeuGc may harm the efficacy and safety of cell therapy products. Sialic acids have been determined by derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB) followed by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS); however, the limited availability of cell therapy products requires more sensitive and specific methods for the quality test. Here we studied the use of nano-flow liquid chromatography/Fourier transformation ion cyclotron resonance mass spectrometry (nanoLC/FTMS) and nanoLC/MS/MS for NeuGc-specific determination at a low femtomole level. Using our method, we found NeuGc contamination of the human cell line (HL-60RG cells) cultured with human serum. Our method needs only 2.5x10(3) cells for one injection and would be applicable to the determination of NeuGc in cell therapy products.

DOI： 10.1016/j.chroma.2007.05.062

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Language：English   Publisher：OXFORD UNIV PRESS INC  

DOI： 10.1093/glycob/cwm041

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.

DOI： 10.1093/glycob/cwl086

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FEDERATION AMER SOC EXP BIOL  

Jacalin, an (alpha-O-glycoside of the disaccharide Thomsen-Friedenreich antigen (galactose (beta 1-3 N-acetylgalactosamine, T-antigen)-specific lectin from jackfruit seeds, has been shown to induce mitogenic responses and to block infection by HIV-1 in CD4(+) T lymphocytes. The molecular mechanism underlying Jacalin-induced T cell activation has not been elucidated completely yet. In the present study, protein tyrosine phosphatase (PTPase) CD45 was isolated from a Jurkat T cell membrane fraction as a major receptor for Jacalin through affinity chromatography and mass spectrometry. CD45, which is highly glycosylated and expressed exclusively on the surface of lymphocytes, is a key regulator of lymphocyte signaling, playing a pivotal role in activation and development. We found that the lectin induced significant IL-2 production by a CD45-positive Jurkat T cell line (JE6.1) and primary T cells. However, this effect did not occur in a CD45-negative Jurkat T cell line (J45.01) and was blocked completely by a specific CD45 PTPase inhibitor in Jurkat T (JE6.1) and primary T cells. Furthermore, we also observed that Jacalin caused a marked increase in IL-2 secretion in response to TCR ligation and CD28 costimulation and contributed to Th1/Th2 cytokine production by activating CD45. Jacalin increased CD45 tyrosine phosphatase activity, which resulted in activation of the ERK1/2 and p38 MAPK cascades. Based on these findings, we propose a new, immunoregulatory model for Jacalin, wherein glycosylation-dependent interactions of Jacalin with CD45 on T cells elevate TCR-mediated signaling, which thereby up-regulate T cell activation thresholds and Th1/Th2 cytokine secretion.

DOI： 10.1189/jlb.1106660

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The core fucosylation (alpha 1,6-fucosylation) of glycoprotein is widely distributed in mammalian tissues. Recently alpha 1,6-fucosylation has been further reported to be very crucial by the study of alpha 1,6-fucosyltransferase (Fut8)-knock-out mice, which shows the phenotype of emphysema-like changes in the lung and severe growth retardation. In this study, we extensively investigated the effect of core fucosylation on alpha 3 beta 1 integrin and found for the first time that Fut8 makes an important contribution to the functions of this integrin. The role of core fucosylation in alpha 3 beta 1 integrin-mediated events has been studied by using Fut8(+/+) and Fut8(-/-) embryonic fibroblasts, respectively. We found that the core fucosylation of alpha 3 beta 1 integrin, the major receptor for laminin 5, was abundant in Fut8(+/+) cells but was totally abolished in Fut8(-/-) cells, which was associated with the deficient migration mediated by alpha 3 beta 1 integrin in Fut8(-/-) cells. Moreover integrin-mediated cell signaling was reduced in Fut8(-/-) cells. The reintroduction of Fut8 potentially restored laminin 5-induced migration and intracellular signaling. Collectively, these results suggested that core fucosylation is essential for the functions of alpha 3 beta 1 integrin.

DOI： 10.1074/jbc.M608764200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of beta 1,6-GlcNAc branching of N-glycans, which contributes to metastasis. N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulting in the suppression of metastasis. It has long been hypothesized that the suppression of GnT-V product formation by the action of GnT-III would also exist in vivo, which will consequently lead to the inhibition of biological functions of GnT-V. To test this, we draw a comparison among MKN45 cells, which were transfected with GnT-III, GnT-V, or both, respectively. We found that alpha 3 beta 1 integrin-mediated cell migration on laminin 5 was greatly enhanced in the case of GnT-V transfectant. This enhanced cell migration was significantly blocked after the introduction of GnT-III. Consistently, an increase in bisected GlcNAc but a decrease in beta 1,6-GlcNAc-branched N-glycans on integrin alpha 3 subunit was observed in the double transfectants of GnT-III and GnT-V. Conversely, GnT-III knockdown resulted in increased migration on laminin 5, concomitant with an increase in beta 1,6-GlcNAc-branched N-glycans on the alpha 3 subunit in CHP134 cells, a human neuroblastoma cell line. Therefore, in this study, the priority of GnT-III for the modification of the alpha 3 subunit may be an explanation for why GnT-III inhibits GnT-V-induced cell migration. Taken together, our results demonstrate for the first time that GnT-III and GnT-V can competitively modify the same target glycoprotein and furthermore positively or negatively regulate its biological functions.

DOI： 10.1074/jbc.M607274200

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Language：Japanese   Publisher：(一財)医薬品医療機器レギュラトリーサイエンス財団  

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Ceruloplasmin has ferroxidase activity and plays an essential role in iron metabolism. In this study, a site-specific glycosylation analysis of human ceruloplasmin (CP) was carried out using reversed-phase high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). A tryptic digest of carboxymethylated CP was subjected to LC-ESI-MS/MS. Product ion spectra acquired data-dependently were used for both distinction of the glycopeptides from the peptides using the carbohydrate B-ions, such as m/z 204 (HexNAc) and m/z 366 (HexHexNAc), and identification of the peptide moiety of the glycopeptide based on the presence of the b- and y-series ions derived from the peptide. Oligosaccharide composition was deduced from the molecular weight calculated from the observed mass of the glycopeptide and theoretical mass of the peptide. Of the seven potential N-glycosylation sites, four (Asn119, Asn339, Asn378, and Asn743) were occupied by a sialylated biantennary or triantennary oligosaccharide with fucose residues (0, 1, or 2). A small amount of sialylated tetraantennary oligosaccharide was detected. Exoglycosidase digestion suggested that fucose residues were linked to reducing end GlcNAc in biantennary oligosaccharides and to reducing end and/or alpha1-3 to outer arms GlcNAc in triantennary oligosaccharides and that roughly one of the antennas in triantennary oligosaccharides was alpha2-3 sialylated and occasionally alpha1-3 fucosylated at GlcNAc.

DOI： 10.1016/j.ab.2005.10.036

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We have previously described the site-specific glycosylation analysis of rat brain Thy-1 by LC/multistage tandem mass spectrometry (MS') using proteinase-digested Thy-1. In the present study, detailed structures of oligosaccharides released from Thy-1 were elucidated by mass spectrometric oligosaccharide profiling using LC/MS with a graphitized carbon column (GCC-LC/MS). First, using model oligosaccharides, we improved the oligosaccharide profiling by ion trap mass spectrometry (IT-NIS) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Sequential scanning of a full MSn scan with FT-ICR-MS followed by data-dependent MS' with IT-MS in positive ion mode, and a subsequent full MS1 scan with FT-ICR-MS followed by data-dependent MSn with IT-MS in negative ion mode enabled the monosaccharide composition analysis as well as profiling and sequencing of both neutral and acidic oligosaccharides in a single analysis. The improved oligosaccharide profiling was applied to elucidation of N-linked oligosaccharides from Thy-1 isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was demonstrated that Thy-1 possesses a significant variety of N-linked oligosaccharides, including Lewis a/x, Lewis b/y, and disialylated structure as a partial structure. Our method could be applicable to analysis of a small abundance of glycoproteins, and could become a powerful tool for glycoproteomics. (c) 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.chroma.2005.11.043

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：18  

The alteration of glycosyltransferase expression and the subsequent changes in oligosaccharide structures are reported in several diseases. The analysis of glycan structural alteration in glycoproteins is becoming increasingly important in the discovery of therapies and diagnostic markers. In this study, we propose a strategy for glycomic/glycoproteomic analysis based on oligosaccharide profiling by LC/MS followed by proteomic approaches, including 2-DE and 2-D lectin blot. As a model of aberrant cells, we used Chinese hamster ovary cells transfected with N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the addition of a bisecting N-acetylglucosamine (GlcNAc) to beta-mannose of the mannosyl core of N-linked oligosaccharides. LC/MS equipped with a graphitized carbon column (GCC) enabled us to elucidate the structural alteration induced by the GnT-III expression. Using 2-D lectin blot followed by LC/MS/MS, the protein carrying an extra N-acetylhexosamine in cells transfected with GnT-III was successfully identified as integrin alpha3. Thus, oligosaccharide profiling by GCC-LC/MS followed by proteomic methods can be a powerful tool for glycomic/glycoproteomic analysis.

DOI： 10.1002/pmic.200401330

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Language：Japanese  

DOI： 10.14921/jscc1971b.34.4_309

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We developed an efficient and convenient strategy for protein identification and glycosylation analysis of a small amount of unknown glycoprotein in a biological sample. The procedure involves isolation of proteins by electrophoresis and mass spectrometric peptide/glycopeptide mapping by LC/ion trap mass spectrometer. For the complete glycosylation analysis, proteins were extracted in intact form from the gel, and proteinase-digested glycoproteins were then subjected to LC/multistage tandem MS (MSn) incorporating a full mass scan, in-source collision-induced dissociation (CID), and data-dependent MSn. The glycopeptides were localized in the peptide/glycopeptide map by using oxonium ions such as HexNAc(+) and NeuAc(+), generated by in-source CID, and neutral loss by CID-MS/MS. We conducted the search analysis for the glycopeptide identification using search parameters containing a possible glycosylation at the Asn residue with N-acetylglucosamine (203 Da). We were able to identify the glycopeptides resulting from predictable digestion with proteinase. The glycopeptides caused by irregular cleavages were not identified by the database search analysis, but their elution positions were localized using oxonium ions produced by in-source CID, and neutral loss by the data-dependent MSn. Then, all glycopeptides could be identified based on the product ion spectra which were sorted from data-dependent CID-MSn spectra acquired around localized positions. Using this strategy, we successfully elucidated site-specific glycosylation of Thy-1, glycosylphosphatidylinositol (GPI)-anchored proteins glycosylated at Asn23, 74, and 98, and at Cys 111. High-mannose- type, complex-type, and hybrid-type oligosaccharides were all found to be attached to Asn23, 74 and 98. and four GPI structures could be characterized. Our method is simple, rapid and useful for the characterization of unknown glycoproteins in a complex mixture of proteins. (c) 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.chroma.2005.07.100

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Mannan-binding protein (MBP) is a C-type serum lectin that is known to be a host defense factor involved in innate immunity, and recognizes mannose, fucose, and N-acetylglucosamine residues. Although some exogenous MBP ligands have been reported, little is known about its endogenous ligands. In the present study, we found that endogenous MBP ligands are highly expressed in the brush border epithelial cells of kidney-proximal tubules by immunohistochemistry, and both meprin α and β (meprins), as novel endogenous MBP ligands, have been identified through affinity chromatography and mass spectrometry. Meprins are membrane-bound and secreted zinc metalloproteases extensively glycosylated and highly expressed in kidney and small intestinal epithelial cells, leukocytes, and certain cancer cells. Meprins are capable of cleaving growth factors, extracellular matrix proteins, and biologically active peptides. Deglycosylation experiments indicated that the MBP ligands on meprins are high mannose- or complex-type N-glycans. The interaction of MBP with meprins resulted in significant decreases in the proteolytic activity and matrix-degrading ability of meprins. Our results suggest that core N-linked oligosaccharides on meprins are associated with the optimal enzymatic activity and that MBP is an important regulator for modulation of the localized meprin proteolytic activity via N-glycan binding. Because meprins are known to be some of the major matrix-degrading metalloproteases in the kidney and intestine, MBP, which functions as a natural and effective inhibitor of meprins, may contribute, as a potential therapeutic target, to tumor progression by facilitating the migration, intravasation, and metastasis of carcinoma cells, and to acute renal failure and inflammatory bowel diseases. Copyright © 2005 by The American Association of Immunologists, Inc.

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The HNK-1 carbohydrate, which is recognized by anti-HNK-1 antibody, is well known to be expressed predominantly in the nervous system. The characteristic structural feature of the HNK-1 carbohydrate is 3-sulfoglucuronyl residues attached to lactosamine structures (Galβ1-4GlcNAc) on glycoproteins and glycolipids. The biosynthesis of the HNK-1 carbohydrate is regulated mainly by two glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase. In this study, we found that GlcAT-S mRNA was expressed at higher levels in the kidney than in the brain, but that both GlcAT-P and HNK-1 sulfotransferase mRNAs, which were expressed at high levels in the brain, were not detected in the kidney. These results suggested that the HNK-1 carbohydrate without sulfate (non-sulfated HNK-1 carbohydrate) is expressed in the kidney. We substantiated this hypothesis using two different monoclonal antibodies: one (anti-HNK-1 antibody) requires sulfate on glucuronyl residues for its binding, and the other (antibody M6749) does not. Western blot analyses of mouse kidney revealed that two major bands (80 and 140 kDa) were detected with antibody M6749, but not with anti-HNK-1 antibody. The 80- and 140-kDa band materials were identified as meprin α and CD13/aminopeptidase N, respectively. We also confirmed the presence of the non-sulfated HNK-1 carbohydrate on N-linked oligosaccharides by multistage tandem mass spectrometry. Immunofluorescence staining with antibody M6749 revealed that the non-sulfated HNK-1 carbohydrate was expressed predominantly on the apical membranes of the proximal tubules in the cortex and was also detected in the thin ascending limb in the inner medulla. This is the first study indicating the presence of the non-sulfated HNK-1 carbohydrate being synthesized by GlcAT-S in the kidney. The results presented here constitute novel knowledge concerning the function of the HNK-1 carbohydrate. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M501728200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We have previously demonstrated that liquid chromatography/mass spectrometry equipped with a graphitized carbon column (GCCLC/MS) is useful for the structural analysis of carbohydrates in a glycoprotein. Here, we studied the monosaccharide composition analysis and quantitative oligosaccharide profiling by GCC-LC/MS. Monosaccharides were labeled with 2-aminopyridine and then separated and monitored by GCC-LC/MS in the selective ion mode. The use of tetradeuterium-labeled pyridylamino (d(4)-PA) monosaccharides as internal standards, which were prepared by the tagging of standard monosaccharides with hexadeuterium-labeled 2-aminopyridine (d(6)-AP), afforded a good linearity and reproducibility in ESIMS analysis. This method was successfully applied to the monosaccharide composition analysis of model glycoproteins, fetuin, and erythropoietin. For quantitative oligosaccharide profiling, oligosaccharides released from an analyte and a standard glycoprotein were tagged with do- and d(6)-AP, respectively, and an equal amount of do- and d(4)-PA oligosaccharides were coinjected into GCC-LC/MS. In this procedure, the oligosaccharides that existed in either analyte or a standard glycoprotein appeared as single ions, and the oligosaccharides that existed in both analyte and a standard glycoprotein were detected as paired ions. The relative amount of analyte oligosaccharides could be determined on the basis of the analyte/internal standard ion-pair intensity ratio. The quantitative oligosaccharide profiling enabled us to make a quantitative and qualitative comparison of glycosylation between the analyte and standard glycoproteins. The isotope tag method can be applicable for quality control and comparability assessment of glycoprotein products as well as the analysis of glycan alteration in some diseases. (c) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.chroma.2004.11.070

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Aim: To develop a rapid and sensitive method for monosaccharide composition analysis. Methods: Glycoprotein was first hydrolyzed to monosaccharides, which were subsequently reacetylated (amino monosaccharides), tagged with 2-aminopyridine and then separated and monitored in selected ion mode by CapGCC-LC/MS. The use of tetradeuterium labeled-aminopyridyl-monosaccharides prepared by tagging monosacahrides with hexadeuterium labeled 2-aminopyridine as internal standards improved the linearity and reproducibility in quantification. Results: This method was successfully applied to monosaccharide composition analysis of model glycoproteins, fetuin and erythropoietin down to 1 pmol monosaccharides. Conclusion: This method has been shown to be highly sensitive and is applicable to monosaccharide composition analysis of glycoproteins.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS LTD  

The Lewis x structure [Le(x), Gal beta 1-4(Fuc alpha 1-3)GlcNAc] motif is one of the tumor antigens and plays an important role in oncogenesis, development, cellular differentiation and adhesion. The detection of Le(x)-carbohydrates and their structural analysis are necessary to clarify the role of Le(x) in several biological events. Mass spectrometry has been preferably used for the structural analysis of carbohydrates. Especially, collision-induced dissociation (CID) tandem mass spectrometry (MS/MS), which causes a glycosidic bond cleavage, is used for carbohydrate sequencing. However, Le(x) cannot be identified by MS/MS due to the existence of the positional isomers, such as Lewis a [Gal beta 1-3(alpha 1-4Fuc)GlcNAc]. In the present study, we demonstrate the specific detection of Lex-carbohydrates in a biological sample by using multiple-stage MS/MS (MS'). Using pyridylaminated oligosaccharides bearing Lex, we found that the Le(x)-motif yields a cross-ring fragment by the cleavage of a bond between C-3 and C-4 of GlcNAc in Gal(Fuc)GlcNAc. The Le(x)-specific cross-ring fragment ion at m/z 259 was effectively detected by sequential scans, consisting of a full MS' scan, data-dependent CID MS2 scan, MS3 of [Gal(Fuc)GlcNAc+Na](+) at m/z 534, and MS4 of [GalGlcNAc+Na](+) at m/z 388. The sequential scan was applied to N-linked oligosaccharide profiling using a LC/ESI-MSn system equipped with a graphitized carbon column. We successfully detected the Le(x)-motif and elucidated the structures of several Le(x) and Lewis y [(Fuc alpha 1-2)Gal beta 1-4(Fuc alpha 1-3)GlcNAc] oligosaccharides in the murine kidney used as a model tissue. Our method is expected to be a powerful tool for the specific detection of the Lex-motif, and structural elucidation of Le(x)-carbohydrates in biological samples. Copyright) 2005 John Wiley & Sons, Ltd.

DOI： 10.1002/rcm.2190

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Human apolipoprotein B100 (apoB100) has 19 potential N-glycosylation sites, and 16 asparagine residues were reported to be occupied by high-inannose type, hybrid type, and monoantennary and biantennary complex type oligosaccharides. In the present study, a site-specific glycosylation analysis of apoB100 was carried out using reversed-phase high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI MS/ MS). ApoB100 was reduced, carboxymethylated, and then digested by trypsin or chymotrypsin. The complex mixture of peptides and glycopeptides was subjected to LC/ESI MS/ MS, where product ion spectra of the molecular ions were acquired data-dependently. The glycopeptide ions were extracted and confirmed by the presence of carbohydrate-specific fragment ions, such as mlz 204 (HexNAc) and 366 (HexHexNAc), in the product ion spectra. The peptide moiety of glycopeptide was determined by the presence of the b- and y-series ions derived from its amino acid sequence in the product ion spectrum, and the oligosaccharide moiety was deduced from the calculated molecular mass of the oligosaccharide. The heterogeneity of carbohydrate structures at 17 glycosylation sites was determined using this methodology. Our data showed that Asn2212, not previously identified as a site of glycosylation, could be glycosylated. It was also revealed that Asn158, 1341, 1350, 3309, and 3331 were occupied by high-mannose type oligosaccharides, and Asn 956, 1496, 2212, 2752, 2955, 3074, 3197, 3438, 3868, 4210, and 4404 were predominantly occupied by mono- or disialylated oligosaccharides. Asn3384, the nearest N-glycosylation site to the LDL-receptor binding site (amino acids 3359-3369), was occupied by a variety of oligosaccharides, including high-mannose, hybrid, and complex types. These results are useful for understanding the structure of LDL particles and oligosaccharide function in LDL-receptor ligand binding. © Oxford University Press 2004
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DOI： 10.1093/glycob/cwi033

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Background: The allergenic potential of chicken egg white ovomucoid (OVM) is thought to depend on its stability to heat treatment and digestion. Pepsin-digested fragments have been speculated to continue to exert an allergenic potential. OVM was digested in simulated gastric fluid (SGF) to examine the reactivity of the resulting fragments to IgE in sera from allergic patients. Methods: OVM was digested in SGF and subjected to SDS-PAGE. The detected fragments were then subjected to N-terminal sequencing and liquid chromatography/mass spectrometry/mass spectrometry analysis to confirm the cleavage sites and partial amino acid sequences. The reactivity of the fragments to IgE antibodies in serum samples from patients allergic to egg white was then determined using Western blotting (n = 24). Results: The rate of OVM digestion depended on the pepsin/OVM ratio in the SGF. OVM was first cleaved near the end of the first domain, and the resulting fragments were then further digested into smaller fragments. In the Western blot analysis, 93% of the OVM-reactive sera also bound to the 23.5- to 28.5-kDa fragments, and 21% reacted with the smaller 7- and 4.5-kDa fragments. Conclusion: When the digestion of OVM in SGF was kinetically analyzed, 21% of the examined patients retained their IgE-binding capacity to the small 4.5-kDa fragment. Patients with a positive reaction to this small peptide fragment were thought to be unlikely to outgrow their egg white allergy. The combination of SGF-digestibility studies and human IgE-binding experiments seems to be useful for the elucidation and diagnosis of the allergenic potential of OVM. Copyright © 2005 S. Karger AG, Basel.

DOI： 10.1159/000082581

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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. © 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.chroma.2004.08.049

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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. © 2004 Pharmaceutical Society of Japan.

DOI： 10.1248/bpb.27.781

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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. © 2004 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biologicals.2004.04.001

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Other Link： http://search.jamas.or.jp/link/ui/2005215652

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

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 α-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. © 2003 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0003-2697(03)00031-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

The presence of replication-competent adenovirus (RCA) in clinical lots of adenovirus vectors raises a variety of safety concerns. To detect RCA in adenovirus vector products, the cell culture/cytopathic effect (CPE) method has generally been preferred. However, it is difficult to evaluate the amount of RCA clearly and quantitatively by this method. In addition, the cell culture/CPE method requires large-scale cell culturing and a substantial amount of time. For the purpose of establishing a method to detect RCA more sensitively and rapidly, we developed the infectivity PCR, a hybrid method that combines the infectivity assay and quantitative PCR. This method allows RCA to be quantified by real-time quantitative PCR using primers and a probe designed for E1 DNA. By infectivity PCR, 1 pfu of RCA spiked into 109 particles of adenovirus vectors could be detected. In contrast, CPE was observed in the cells infected with 104 pfu of RCA spiked into 109 particles of adenovirus vectors. The glass-beads method was suitable for extracting DNA rapidly from the RCA-infected cells. These results showed that infectivity PCR combined with the glass-beads-based DNA extraction method was useful for the detection of RCA in adenovirus vector products.

DOI： 10.1016/j.ymthe.2003.09.001

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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 NeuAc0-2, 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 β-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. © 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0021-9673(02)01423-1

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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. © 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0021-9673(02)00951-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press  

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. © 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved.

DOI： 10.1006/biol.2002.0339

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press  

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. © 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved.

DOI： 10.1006/biol.2002.0324

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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.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

We previously demonstrated that high-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column (GCC) is useful for the structural analysis of carbohydrates in glycoproteins. Using LC/MS with GCC, sulfated N-linked oligosaccharides were found in erythropoietin (EPO) expressed in baby hamster kidney cells. Sulfation occurs in a part of the N-linked oligosaccharides in the EPO. Sulfated monosaccharide residue in the sulfated N-linked oligosaccharide was determined by exoglycosidase digestion followed by sugar mapping by LC/MS. The linkage position and branch-location of the sulfate group in the tetraantennary oligosaccharide were analyzed by H-1-nuclear magnetic resonance. It was suggested that sulfation occurs on the C-6 position of GlcNAc located in the GlcNAcbeta1-4Manalpha1-3 branch.

DOI： 10.1093/glycob/11.12.1043

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We previously reported that ganglioside GD1a, which is highly expressed in poorly metastatic FBJ-S1 cells, inhibits the serum-induced motility of FBJ-LL cells and that the metastatic potential of FBJ-LL cells is completely suppressed by enforced GD1a expression (Hyuga et al., Int J Cancer 1999
83:685-91). We recently discovered that hepatocyte growth factor (HGF) induces FBJ-LL cell motility. In the present study, the HGF-induced motility of FBJ-S1 cells was found to be one-thirtieth that of FBJ-LL cells. This motility of GD1a-expressing transfectants, which were produced by transfection of FBJ-LL cells with GM2/GD2 synthase cDNA, decreased with increases in their GD1a expression and HGF induced almost no motility in GD1a-pretreated FBJ-LL cells, indicating that GD1a inhibits the HGF-induced motility of FBJ-LL cells. The expression of the HGF receptor c-Met on FBJ-S1 cells, FBJ-LL cells, transfectants and a mock-transfectant was almost the same. The level of tyrosine phosphorylation of c-Met after HGF stimulation in FBJ-S1 cells, GD1a-pretreated FBJ-LL cells and a GD1a-expressing transfectant was significantly lower than in FBJ-LL cells and a mock-transfectant. These findings suggested that GD1a inhibits the HGF-induced motility of FBJ-LL cells through suppression of tyrosine phosphorylation of c-Met. HepG2 cells, a human hepatoma cell line, were used to investigate whether GD1a interferes with other cancer cells expressing c-Met. HepG2 cells did not express GD1a. HGF induced cell scattering of HepG2 cells and the scattering was inhibited by pretreating the cells with GD1a. The c-Met in the cells was autophosphorylated by stimulation with HGF, but after treating the cells with GD1a, the HGF-induced autophosphorylation of c-Met was suppressed. These results suggest that GD1a acts as a negative regulator of c-Met in cancer cells. © 2001 Wiley-Liss, Inc.

DOI： 10.1002/ijc.1481

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Selective glycopeptide mapping of recombinant human erythropoietin (rhEPO) used as a model glycoprotein was successfully carried out by on-line high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) using a Vydac C18 column eluted in acetonitrile-1 mM ammonium acetate, pH 6.8. rhEPO expressed in a Chinese hamster ovary clone was exhaustively digested into four glycopeptides and nine peptides with endoproteinase Glu-C. Both glycopeptides and peptides were eluted with trifluoroacetic acid as the eluent, whereas only glycopeptides were eluted selectively with ammonium acetate in the following order: N38, N24, O126, and N83. Furthermore, many glycoforms included in each glycopeptide were found to be separated by differences in the numbers of sialic acid and N-acetyllactosaminyl repeats. Twenty, 16 and 22 different N-linked oligosaccharides were determined at Asn24, 38, and 83, respectively, and two different O-linked oligosaccharides were observed at Ser126. Our method is simple, rapid, and useful for determining the carbohydrate structures at each glycosylation site and for elucidating the site-specific carbohydrate heterogeneity. © 2001 Elsevier Science B.V.

DOI： 10.1016/S0021-9673(00)01116-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

High-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) and liquid chromatography with tandem mass spectrometry (LC/MS/MS) were applied to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin (EPO) used as a model of the sialylated glycoprotein. N-linked oligosaccharides were released from recombinant human EPO expressed in Chinese hamster ovary cells enzymatically and reduced with NaBH4. Many different sialylated oligosaccharides of EPO were separated and characterized by LC/MS equipped with a graphitized carbon column (GCC). Glycosylation sites and the preliminary glycosylation pattern at each glycosylation site were determined by LC/MS of endoproteinase Glu-C-digested EPO. The detailed site-specific carbohydrate heterogeneity caused by the differences in the molecular weight, branch, linkage, and sequence was elucidated by GCC-LC/MS of the N-linked oligosaccharides released from the isolated glycopeptides. Structural details of the isomers were analyzed by LC/MS/MS, and it was indicated that di- and trisialylated tetraantennary oligosaccharides are attached to Asn24, 38, and 83, whereas their isomers, di- and trisialylated triantennary oligosaccharides containing N-acetyllactosamines, are combined with Asn24. Our method is useful for the determination of glycosylation sites, the site-specific carbohydrate heterogeneity of glycoproteins, and the carbohydrate structure. (C) 2000 Academic Press.

DOI： 10.1006/abio.2000.4739

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Crocus sativus lectin (CSL) is one of the truly mannose-specific plant lectins that has a unique binding specificity that sets it apart from others. We studied sugar-binding specificity of CSL in detail by a solution phase method (fluorescence polarization) and three solid phase methods (flow injection, surface plasmon resonance, and microtiter plate), using a number of different glycopeptides and oligosaccharides. CSL binds the branched mannotriose structure in the N-glycan core. Substitution of the terminal Man in the Manα(1-3)Man branch with GlcNAc drastically decreases binding affinity much more than masking of the terminal Man in the Manα(1-6)Man branch. Most interestingly, the β-Man-linked GlcNAc in N-glycan core structure contributes greatly to the binding. The effect of this GlcNAc is so strong that it can substantially offset the negative effect of substitution on the nonreducing terminal Man residues. On the other hand, the GlcNAc that is usually attached to Asn in N-glycans and the L-Fuc linked at the 6-position of the GlcNAc are irrelevant to the binding. A bisecting GlcNAc neither contributes to nor interferes with the binding. This unique binding specificity of CSL offers many possibilities of its use in analytical and preparative applications.

DOI： 10.1074/jbc.M003765200

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We demonstrate the difference between the follistatin isoforms (FS-288 and FS-315), two activin-binding proteins, in the neutralizing activity for activin signalling. Transcriptional reporter assay using 3TP-Lux, an activin- responsive reporter construct, showed that the inhibitory effect of FS-288 on activin-induced transcriptional response is more potent than that of FS-315. The potency was not influenced by the presence of heparan sulfates, by which FS, in particular FS-288, associates with cell surfaces at a high affinity. Furthermore, FS-288 inhibited the binding of activin to its type II receptor more markedly than did FS-315, as evidenced by surface plasmon resonance and affinity cross-linking experiments. Moreover, the Kd of FS-288 and FS-315 for activin A was estimated to be 46.5 ± 0.37 pM and 432 ± 26 pM, respectively, by surface plasmon resonance experiments. These results indicate that the different potency between the two FS isoforms in the inhibition of activin activities depends on their affinity for activin A. (C) 2000 Elsevier Science Inc.

DOI： 10.1016/S0898-6568(00)00099-1

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The molecular mechanism of organ-specific metastasis to the liver remains largely unknown. However, it is conceivable that paracrine growth factors produced by a target organ induce migration and proliferation of malignant cells to that organ, and this is the cause of organ-specific metastasis. In this study, we investigated the effect of hepatocyte growth factor/scatter factor (HGF/SF) and activin A, which are known to be produced by the liver, on the motility and growth of liver-metastatic cell line FBJ-LL. HGF/SF and activin A induced motility synergistically, but they did not affect the proliferation of FBJ-LL cells. Expression of the HGF/SF receptor, the c-met gene, and the activin-receptor type IA, type IB, and type IIA genes in FBJ-LL cells was detected by reverse transcription polymerase chain reaction. These findings suggest that both HGF/SF and activin A promote organ-specific metastasis to the liver by induction of migration through their specific receptors on liver-metastatic cells. Copyright (C) 2000 Elsevier Science Ireland Ltd.

DOI： 10.1016/S0304-3835(00)00360-8

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Comparative studies concerning glycoform analysis of sialoglycoproteins by capillary electrophoresis were performed using a few separation modes hitherto reported. Glycoprotein samples examined in the present study were successfully separated to their respective glycoforms using surface-modified capillaries commercially available for capillary gas chromatography in the running buffer near their isoelectric points. The analysis times were less than 50 min and reproducibilities in migration times were excellent (less than 2.0% RSD for both run-to-run and day-to-day analyses). We present a method for the glycoform analysis of α1-acid glycoprotein in sera by simple pre-treatment as an application. The present technique will become one of the general methods for the evaluation of glycosylation heterogeneity of commercially available glycoprotein drugs. Copyright (C) 2000 Elsevier Science B.V.

DOI： 10.1016/S0021-9673(99)01080-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

DOI： 10.1006/abio.1999.4077

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

High-performance liquid chromatography with online electrospray ionization mass spectrometry (ESI-LC/MS) was investigated for the analysis of carbohydrate heterogeneity using RNase B as a model glycoprotein. Oligosaccharides released from RNase B with endoglycosidase H were reduced and separated on a graphitized carbon column (GCC). GCC-HPLC/MS in the positive-ion mode was successful in the identification of one Man5GlcNAc, three Man6GlcNAc, three Man7GlcNAc, three Man8GlcNAc, one Man9GlcNAc, and an oligosaccharide having six hexose units (Hex) and two N-acetylhexosamine units (HexNAc). The branch structures of the three Man7GlcNAc isomers were determined by liquid chromatography with tandem mass spectrometry (LC/MS/MS). LC/MS/MS analysis was shown to be useful for the detection and identification of a trace amount of Hex6HexNAc2 alditol as a hybrid-type oligosaceharide. Its structure was confirmed by the combination of LC/MS with enzymatic digestion using β-galactosidase and N-acetyl-β-glucosaminidase. The relative quantities of high-mannose-type oligosaccharides in RNase B detected by ESI-LC/MS are in reasonable agreement with those by UV, high-pH anion- exchange chromatography with pulsed amperometric detection, fluorophore- assisted carbohydrate electrophoresis. Our results indicate that LC/MS and LC/MS/MS can be utilized to elucidate the distribution of oligosaccharides and their structures, which differ in molecular weight, sugar sequence, and branch structure.

DOI： 10.1006/abio.1999.4026

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We have demonstrated that iron controls hemoglobin (Hb) synthesis in erythroid differentiating K562 cells by enhancing the activity of a key enzyme of the Hb synthesis, δ-aminolevulinate synthase (ALAS). In the present study, we studied iron mobilization and the role of iron in erythroid differentiating cells by measuring the level of iron by means of high-performance liquid chromatography using electrochemical detection (HPLC-ED). After treatment of K562 cells with sodium butyrate, the expression of transferrin receptor (TfR) increased initially, followed by an increase in the levels of both total iron and Hb as well as the ALAS activity. However, no increase could be found in the levels of non-heme iron, low-molecular-mass iron (LMMFe) and ferritin. Addition of diferric transferrin (FeTf) enhanced both δ-aminolevulinic acid (ALA) and Hb synthesis. In contrast, addition of hemin elevated the levels of all iron species as well as the Hb synthesis but reduced the TfR expression and ALA contents in both butyrate treated and untreated cells. These results suggest that Hb synthesis is controlled by TfR expression, and that the ALA synthesis is suppressed by iron released from heme and/or Hb due to lowered expression of TfR.

DOI： 10.1016/S0378-4347(97)00511-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press  

Quantum dye (QD), a macrocyclic europium-chelate, developed as a cytological marker, has never been used for quantitative applications. It would be ideal, however, if the same tracer can be used for both qualitative and quantitative purposes. We have labeled some lectins and neoglycoproteins with QD for the purpose of quantitative analyses in glycobiology: and tested its suitability in three different areas in glycobiology: (1) glycosyltransferase, (2) an animal lectin mannose-binding protein, and (3) the Gal/GalNAc receptor of rat liver membrane. Usefulness of QD-labeled lectins was amply demonstrated by the quantification of galactosyltransferase activity using QD-soybean agglutinin and QD-RCA120 (Ricinus communis agglutinin). We also showed that QD-labeled neoglycoproteins, QD-Man-BSA and QD-Gal-BSA, BSA, can replace radioiodinated counterparts in the binding assays of animal lectins (serum mannose binding protein and hepatic Gal/GalNAc receptor.) The advantage of QD and other europium labels is that it does not decay as radioiodides do. The long shelf-life results in more consistent results from repeated experiments.

DOI： 10.1093/glycob/8.9.849

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DOI： 10.1006/abio.1997.2233

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Informa Healthcare  

DOI： 10.3109/10715769709065769

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K562 cells were used to investigate the factors that control hemoglobin (Hb) synthesis. Treatment with sodium butyrate enhanced Hb synthesis and glycophorin A expression. δ-Aminolevulinate synthase (ALAS) activity and Hb levels simultaneously increased to a similar extent and with a similar time course, and the increases were dependent on the concentration of diferric transferrin (FeTf) in the culture medium. Addition of exogenous δ-aminolevulinic acid (ALA) resulted in a dose-dependent increase in Hb content. Hb synthesis was inhibited 50% after addition of succinylacetone (SA), a potent inhibitor of δ-aminolevulinate dehydratase. These findings suggest that ALAS is a key enzyme in the eight steps of de novo heme synthesis and that iron, including FeTf, plays a central role in Hb synthesis through control of ALAS activity in erythroid differentiating cells. On the other hand, erythropoietin (EPO) treatment had no effect on Hb synthesis and slightly suppressed glycophorin A expression. Hemin enhanced Hb synthesis in the K562 cells but not glycophorin A expression. The addition of ALA, SA, or FeTf to hemin-treated cells caused no significant changes in Hb synthesis. Butyrate, EPO, and hemin acted on the K562 cells in different ways and caused different biochemical changes in the Hb synthesis process. © 1996 Academic Press, Inc.

DOI： 10.1006/abbi.1996.0175

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The effects of acetaminophen (AA) on the ultrastructure of isolated hepatocytes (IHC) of rat following incubation of IHC suspensions with AA were examined by electron microscopy. The effect of N-acetyl-p-benzoquinone imine (NAPQI), a putative toxic metabolite of AA, were also observed. IHC were prepared from livers obtained from phenobarbital-treated rats by collagenase perfusion method. With 5 and 20 mM AA, surface blebs mainly containing smooth endoplasmic reticulum (ER) occurred in IHC. Dilatation of Golgi apparatus, partial degranulation of rough ER and enlargement of mitochondria were also observed. The altered mitochondria showed a low electron-dense matrix with loss of mitochondrial granules. With 500 μM NAPQI, surface blebs containing various organelles occurred in IHC. Disorderly distributions of cytoplasmic organelles, mild dilatation of rough and smooth ER and cytoplasmic myeloid bodies were observed. The characteristic myeloid bodies were seemingly derived from degranulated rough ER. © 1995, All rights reserved.

DOI： 10.1016/S0940-2993(11)80345-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Informa Healthcare  

The effects of active oxygen species on the in vivo activity of recombinant human erythropoietin (EPO) treated by Fenton system, xanthine (X) plus xanthine oxidase (XO) system and hydrogen peroxide (H2O2) has been studied by means of counting the increase in number of hemolyser-resistant cells (HRCs) in EPO-injected mice. The results showed that both Fenton and X plus XO systems caused a significant reduction of the activity in proportion to the concentration of generated active oxygen species. Meanwhile, the treatment of EPO with H2O2 alone resulted in a relatively slight reduction of the activity. Electrophoretic studies on the structure of EPO revealed that its main protein band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) disappeared in proportion with the extent of exposure to active oxygen generating systems. Both Fenton and X plus XO systems caused a significant loss of fluorescence in the pyridylamino (PA-) sugar chain in proportion to the concentration of generated active oxygen species, and no degradation products in the sugar chain part of the PA-sugar chain were detected. This showed that aromatic groups in EPO were sensitive to attack by active oxygen species. These results provide evidence that hydroxyl radical and other active oxygen species have a potential to react with EPO, leading to a reduction of its in vivo activity. © 1995 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

DOI： 10.3109/10715769509147542

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Non-protein-bound iron in human synovial fluid was determined using high-performance liquid chromatography with electrochemical detection. The procedure was based on the separation of the iron-diethylenetriaminepentaacetic acid (DPTA) complex formed directly on a chromatographic column containing an anion-exchange resin followed by electrochemical detection. The method enabled more than 0.1 μM Fe(III) to be determined with an injection volume of 10 μl. A mixture of synovial fluid, 20 μM DTPA and acetate buffer was incubated in the presence and absence of superoxide (O-2) generated by a xanthine-xanthine oxidase system and was ultrafiltered through a 30 000 molecular mass cut-off filter. No iron was detected in the ultrafiltrate at physiological pH. However, the presence of iron was observed in the ultrafiltrate at low pH, and O-2 and decreased pH, iron may be released into the synovial fluid. © 1994.

DOI： 10.1016/0378-4347(94)00135-9

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A new, rapid, sensitive, and specific method combining ion chromatography with electrochemical detection was developed for measuring non-protein-bound Fe(II) and Fe(III) in biological samples. The procedure was based on the separation of the iron-diethylenetri-aminepentaacetic acid complex formed directly on the chromatographic column with anion-exchange resin followed by electrochemical detection. The method enabled more than 0.5 μm Fe(II) and Fe(III) to be determined for injection volumes of 10 μl. This method was applicable for the determination of Fe(II) and Fe(III) in ultrafiltrates of the rat liver cytosolic fraction. It was found that release of iron from iron-bound proteins was pH dependent and that non-protein-bound iron in the tissues was determined in a ferrous state at low pH values. © 1991.

DOI： 10.1016/0003-2697(91)90192-V

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A simple, rapid and sensitive method for the determination of iron(III) ion by ion chromatography coupled with electrochemical detection was developed. The method reduced the interferences of iron(III) ions and enabled more than 5 pmol of iron(III) to be determined with an injection volume of 10 μl. The method was applied to the determination of ferroxidase activity of ceruloplasmin with good reproducibility. The production of iron(III) ion by cerulplasmin was found to be linear with respect to reaction time and protein concentration. © 1990.

DOI： 10.1016/S0021-9673(01)81505-3

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Aldose reductase (alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21) and aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2) were purified to a homogeneity from rat testis. The molecular weights of aldose reductase and aldehyde reductase were estimated to be 38 000 and 41 000 by SDS-polyacrylamide gel electrophoresis, and the pI values of these enzymes were found to be 5.3 and 6.1 by chromatofocusing, respectively. Aldose reductase had activity for aldo-sugars such as xylose, glucose and galactose, whereas aldehyde reductase was virtually inactive for these aldo-sugars. The Km values of aldose reductase for aldo-sugars were relatively high. When a correction was made for the fraction of aldo-sugar present as the aldehyde form, which is the real substrate of the enzyme, the Km values were much lower. Aldose reductase utilized both NADPH and NADH as coenzyme, whereas aldehyde reductase utilized only NADPH. Aldose reductase was activated significantly by sulfate ion, while aldehyde reductase was little affected. Both enzymes were inhibited strongly by the known aldose reductase inhibitors. However, aldehyde reductase was in general less susceptible to these inhibitors when compared to aldose reductase. Both aldose reductase and aldehyde reductase treated with pyridoxal 5-phosphate have lost the susceptibility to aldose reductase inhibitor, suggesting that in these two enzymes aldose reductase inhibitor interacts with a lysine residue. © 1989.

DOI： 10.1016/0167-4838(89)90090-3

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Repeated oral doses of di‐n‐butyl phthalate (DBP) to male rats caused a decrease in testicular fructose and glucose and a sloughing of the germ cells on the first day of treatment. On day 2, more severe sloughing was seen and was accompanied by decreases in testicular iron and zinc levels and increases in the level of inositol and cholesterols. The sloughing was followed by atrophy, accompanied by dissociation of the germ cells from the Sertoli cells and reduction of triglycerides, cholesterols and phospholipids containing choline and ethanolamine residues in the testis. Copyright © 1989 John Wiley &amp
Sons, Ltd.

DOI： 10.1002/jat.2550090413

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これまでに蓄積された科学的知見とリスクマネジメントの考えに基づき、シングルユースシステムを用いて製造されるバイオ医薬品の品質確保に関して考慮すべき要件を提言した。本稿では主に以下の項目で述べた。1)適用範囲、2)基本的要件、3)バイオ医薬品の品質確保及び安定供給のためのリスクアセスメント、4)バイオ医薬品の品質確保及び安定供給のためのリスク対応(管理戦略)、5)ライフサイクルマネジメント。シングルユース製品を用いたバイオ医薬品の製造工程の開発では、シングルユース製品によって生じるバイオ医薬品の品質、有効性、安全性確保及び安定供給に対するリスクとその対応策を明確にし、関係者で共有することが基本的要件となる。

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Language：Japanese   Publisher：(一社)日本薬剤疫学会  

日本で承認されている免疫調節作用を有する抗体医薬品12品目について、報告数の多い有害事象の基本語(Preferred Term;PT)を特定するため、2015年時点で医薬品医療機器総合機構が公開していた医薬品副作用データベースに集積されていた症例を解析した。その結果、報告数の多いPTとして、肺炎、間質性肺疾患、ニューモシスチス・イロベチイ肺炎(Pneumocystiis jiroveci pneumonia;ニューモシスチス肺炎)、蜂巣炎、敗血症、および帯状疱疹等を特定した。これらのPTについて、特に日本で5種類の製品が承認されている抗TNF薬(infliximab、adalimumab、golimumab、certolizumab pegolおよびetanercept)をはじめとした関節リウマチ治療薬に着目し、医薬品ごとに各PTの発現頻度と初回発現時期を解析・比較した。ここでは、シグナル検出指標の報告オッズ比(reporting odds ratio;ROR)を発現頻度の指標として用いた。また、初回発現時期の対照薬としては、抗TNF薬とは標的が異なる関節リウマチ治療薬で、症例の報告数も比較的多い抗インターロイキン-6受容体抗体のtocilizumabを選択した。その結果、肺炎と間質性肺疾患、および敗血症については、RORと初回発現時期の解析からは特定の関節リウマチ治療薬との関連を示唆する結果は得られなかった。一方、ニューモシスチス肺炎については、特にinfliximabで高いRORが得られた。Infliximabではニューモシスチス肺炎の初回発現時期も他の医薬品より早い傾向にあり(0.19yr)、tocilizumab(0.32yr)を対照としてMann-Whitney検定を行うと、有意水準1%で有意差が認められた。Infliximabはニューモシスチス肺炎のRORが高いだけでなく、初回発現時期も特徴的であることから、ニューモシスチス肺炎との間に他よりも強い関連が示唆された。同様なことが、蜂巣炎とtocilizumab、帯状疱疹とcertolizumab pegolについても観察され、感染症に関する一部のPTは、特定のバイオ医薬品に強く関連する可能性が示唆された。肺疾患や皮膚疾患を持つ患者等については、抗TNF薬を使用する際に、これらの感染症が生じやすい可能性に配慮する必要があると考えられた。(著者抄録)

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2017&ichushi_jid=J03595&link_issn=&doc_id=20170428180002&doc_link_id=10.3820%2Fjjpe.21.63&url=https%3A%2F%2Fdoi.org%2F10.3820%2Fjjpe.21.63&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

Language：Japanese  

J-GLOBAL

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Language：Japanese   Publisher：(公社)日本生化学会  

J-GLOBAL

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Language：Japanese  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2016318424

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CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2016109422

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J-GLOBAL

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Language：Japanese   Publishing type：Book review, literature introduction, etc.   Publisher：PHARMACEUTICAL SOC JAPAN  

Therapeutic monoclonal antibodies (mAbs) exert their effects via binding to specific target molecules, which is expected to show rare off-target adverse reactions. However, nonclinical evaluation of mAbs is difficult because they often lack reactivity toward orthologous targets in animals. During the nonclinical evaluation of mAbs, not only the target molecules but fragment crystallizable (Fc) receptors, which regulate the immune effector functions and pharmacokinetic properties of mAbs, should be considered. In this review, factors for extrapolating nonclinical study results to clinical settings are discussed by focusing on Fc receptors. The human Fe gamma receptor family consists of Fc gamma RI, IIa, IIb, IIIa, and IIIb; Fey receptors in laboratory animals are structurally and functionally different from those in humans. In addition, interactions between human IgG-Fc, a component of therapeutic mAbs, and animal Fc gamma receptors are still not fully understood. With regard to neonatal Pc receptor (FcRn), related molecules comprising the FcRn family are not known; however, critical amino acid residues involved in IgG binding are different between human and mouse. In case of next-generation mAbs with a novel structure or mode of action, knowledge from related drugs is limited. To ensure safety of next-generation mAbs, a thorough understanding of the differences in Fc receptors among species and the interactions between mAbs and Fc receptors is required, and the appropriateness of the nonclinical study design should be carefully examined prior to conducting clinical studies.

Web of Science

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：Future Science Ltd  

DOI： 10.4155/bio.15.43

Scopus

PubMed

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

J-GLOBAL

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2015015000

Language：Japanese   Publisher：一般社団法人 日本応用糖質科学会  

DOI： 10.5458/bag.4.3_B39-4

CiNii Books

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Language：Japanese  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2014242643

Language：English   Publisher：日本癌学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

J-GLOBAL

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Language：Japanese   Publisher：(公社)日本生化学会  

J-GLOBAL

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Language：Japanese   Publisher：(公社)日本生化学会  

J-GLOBAL

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Language：Japanese   Publisher：(一社)和漢医薬学会  

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Language：Japanese   Publisher：日本薬剤師会  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2013121965

Language：Japanese  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2014103908

Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

J-GLOBAL

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Language：Japanese   Publisher：日本脂質生化学会  

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Language：Japanese  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2012373603

J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publisher：(一財)医薬品医療機器レギュラトリーサイエンス財団  

蛋白質医薬品の免疫原性を適切に評価することは、後期の段階における開発中止のリスクを軽減し、有効性および安全性に優れた蛋白質医薬品を医療の場に提供する上で重要である。蛋白質医薬品の免疫原性に関連した危険因子、非臨床試験における免疫原性試験の留意点と意義、非臨床試験および臨床試験における蛋白質医薬品の免疫原性の評価戦略、抗体の検出試験と中和活性試験、などについて概説した。

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Language：Japanese  

CiNii Books

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：Japanese  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2011350971

Language：Japanese   Publisher：The Japanese Pharmacological Society  

DOI： 10.1254/fpj.136.280

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2011049115

Language：Japanese   Publisher：(一財)医薬品医療機器レギュラトリーサイエンス財団  

医療現場で使用される治療用蛋白質の75%はヒト型組換え蛋白質で、その殆どはヒト体内で産生される蛋白質と同じアミノ酸配列を有するが、患者で抗体の産生が誘導されたことが報告されている。治療用蛋白質に対する抗体価の測定方法の原理および特徴、抗体活性の測定系構築における留意点、インターフェロン-β、エリスロポエチンおよびパニツムマブに対し患者で産生が誘導された抗体の測定の現状と問題点について概説した。

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Language：Japanese  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2010093272

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS INC  

Web of Science

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Language：Japanese   Publisher：公益社団法人 日本薬学会  

DOI： 10.14894/faruawpsj.45.7_677

CiNii Books

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：GAKUSHIN PUBL CO  

Analysis of the carbohydrate heterogeneity of glycoprotein-based substances is crucial for establishing the nomenclature and definition of biological substances, ensuring consistency in the quality of these products, comparatively assessing the products obtained after the implementation of changes in the manufacturing process, and developing biosimilar or follow-on biological products. Liquid chromatography/mass spectrometry is recognized as one of the most useful techniques for analyzing the carbohydrate heterogeneity of glycoprotein substances. Here, we demonstrate the utility of LC/MS for analyzing the carbohydrate heterogeneity by using some representative glycoproteins such as tissue-plasminogen activator, a monoclonal antibody, the follicle-stimulating hormone, and human chorionic gonadotropin. Further, we demonstrate that MS-based glycoprotein analysis has potential applications in glycomics.

Web of Science

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Language：Japanese   Publisher：公益社団法人 日本薬学会  

DOI： 10.14894/faruawpsj.44.12_1167

CiNii Books

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CiNii Books

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J-GLOBAL

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J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS INC  

Web of Science

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J-GLOBAL

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：Gakushin Publishing Company  

This review presents mass spectrometric methods for glycoprotein identification, determination of glycosylation sites, structural elucidation of carbohydrates, and their applications to glycomics and proteomics. ©2005 FCCA (Forum: Carbohydrates Coming of Age).

DOI： 10.4052/tigg.17.193

Scopus

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Language：English   Publisher：Zoological Society of Japan  

CiNii Books

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Other Link： http://id.nii.ac.jp/1141/00039830/

J-GLOBAL

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Language：Japanese  

CiNii Books

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS INC  

Web of Science

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Language：Japanese   Publisher：日本癌学会  

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese   Publisher：国立医薬品食品衛生研究所化学物質情報部  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2003246017

Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese   Publisher：国立医薬品食品衛生研究所化学物質情報部  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2002188613

Language：Japanese   Publisher：国立医薬品食品衛生研究所化学物質情報部  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2002190543

Language：Japanese   Publisher：日本癌学会  

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese   Publisher：国立医薬品食品衛生研究所化学物質情報部  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/1999087992

Language：English  

CiNii Books

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese   Publisher：(公社)日本薬学会  

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