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Featured researches published by Maho Amano.


FEBS Journal | 2012

Tumour suppressor p16INK4a – anoikis-favouring decrease in N/O-glycan/cell surface sialylation by down-regulation of enzymes in sialic acid biosynthesis in tandem in a pancreatic carcinoma model

Maho Amano; Hanna Eriksson; Joachim C. Manning; Katharina Detjen; Sabine André; Shin-Ichiro Nishimura; Janne Lehtiö; Hans-Joachim Gabius

Tumour suppressor p16INK4a is known to exert cell‐cycle control via cyclin‐dependent kinases. An emerging aspect of its functionality is the orchestrated modulation of N/O‐glycosylation and galectin expression to induce anoikis in human Capan‐1 pancreatic carcinoma cells. Using chemoselective N/O‐glycan enrichment technology (glycoblotting) and product characterization, we first verified a substantial decrease in sialylation. Tests combining genetic (i.e. transfection with α2,6‐sialyltransferase‐specific cDNA) or metabolic (i.e. medium supplementation with N‐acetylmannosamine to track down a bottleneck in sialic acid biosynthesis) engineering with cytofluorometric analysis of lectin binding indicated a role of limited substrate availability, especially for α2,6‐sialylation, which switches off reactivity for anoikis‐triggering homodimeric galectin‐1. Quantitative MS analysis of protein level changes confirmed an enhanced galectin‐1 presence along with an influence on glycosyltransferases (β1,4‐galactosyltransferase‐IV, α2,3‐sialyltransferase‐I) and detected p16INK4a‐dependent down‐regulation of two enzymes in the biosynthesis pathway for sialic acid [i.e. the bifunctional UDP‐N‐acetylglucosamine 2‐epimerase/N‐acetylmannosamine kinase (GNE) and N‐acetylneuraminic acid 9‐phosphate synthase] (P < 0.001). By contrast, quantitative assessment for the presence of nuclear CMP‐N‐acetylneuraminic acid synthase (which is responsible for providing the donor for enzymatic sialylation that also acts as feedback inhibitor of the epimerase activity of GNE) revealed a trend for an increase. Partial restoration of sialylation in GNE‐transfected cells supports the implied role of sialic acid availability for the glycophenotype. Fittingly, the extent of anoikis was reduced in double‐transfected (p16INK4a/GNE) cells. Thus, a second means of modulating cell reactivity to the growth effector galectin‐1 is established in addition to the common route of altering α2,6‐sialyltransferase expression: regulating enzymes of the pathway for sialic acid biosynthesis.


Molecular & Cellular Proteomics | 2010

Sialic Acid-focused Quantitative Mouse Serum Glycoproteomics by Multiple Reaction Monitoring Assay

Masaki Kurogochi; Takahiko Matsushista; Maho Amano; Jun-Ichi Furukawa; Yasuro Shinohara; Masato Aoshima; Shin-Ichiro Nishimura

Despite increasing importance of protein glycosylation, most of the large-scale glycoproteomics have been limited to profiling the sites of N-glycosylation. However, in-depth knowledge of protein glycosylation to uncover functions and their clinical applications requires quantitative glycoproteomics eliciting both peptide and glycan sequences concurrently. Here we describe a novel strategy for the multiplexed quantitative mouse serum glycoproteomics based on a specific chemical ligation, namely, reverse glycoblotting technique, focusing sialic acids and multiple reaction monitoring (MRM). LC-MS/MS analysis of de-glycosylated peptides identified 270 mouse serum peptides (95 glycoproteins) as sialylated glycopeptides, of which 67 glycopeptides were fully characterized by MS/MS analyses in a straightforward manner. We revealed the importance of a fragment ion containing innermost N-acetylglucosamine (GlcNAc) residue as MRM transitions regardless the sequence of the peptides. Versatility of the reverse glycoblotting-assisted MRM assays was demonstrated by quantitative comparison of 25 targeted glycopeptides from 16 proteins between mice with homo and hetero types of diabetes disease model.


Analytical Chemistry | 2010

Glycoblotting-Assisted O-Glycomics: Ammonium Carbamate Allows for Highly Efficient O-Glycan Release from Glycoproteins

Yoshiaki Miura; Kentaro Kato; Yasuhiro Takegawa; Masaki Kurogochi; Jun-ichi Furukawa; Yasuro Shinohara; Noriko Nagahori; Maho Amano; Hiroshi Hinou; Shin-Ichiro Nishimura

Glycoblotting, high throughput method for N-glycan enrichment analysis based on the specific chemical ligation between aminooxy/hydrazide-polymers/solids and reducing N-glycans released from whole serum and cellular glycoproteins, was proved to be feasible for selective enrichment analysis of O-glycans of common (mucin) glycoproteins. We established a standard protocol of glycoblotting-based O-glycomics in combination with nonenzymatic chemical treatment to release reducing O-glycans predominantly from various glycoprotein samples. It was demonstrated that the nonreductive condition employing a simple ammonium salt, ammonium carbamate, made glycoblotting-based enrichment analysis of O-glycans possible without significant loss or unfavorable side reactions. A general workflow of glycoblotting using a hydrazide bead (BlotGlyco H), on-bead chemical manipulations, and subsequent mass spectrometry allowed for rapid O-glycomics of human milk osteopontin (OPN) and urinary MUC1 glycoproteins purified from healthy donors in a quantitative manner. It was revealed that structures of O-glycans in human milk OPN were varied with habitual fucosylation and N-acetyllactosamine units. It was also suggested that purified human urinary MUC1 was modified preferentially by sialylated O-glycans (94% of total) with 7:3 ratio of core 1 to core 2 type O-glycans. Versatility of the present strategy is evident because this method was proved to be suited for the enrichment analysis of general biological and clinical samples such as human serum and urine, cultured human cancer cells, and formalin-fixed paraffin-embedded tissue sections. It is our belief that the present protocols would greatly accelerate discovery of disease-relevant O-glycans as potential biomarkers.


Molecular & Cellular Proteomics | 2010

Threshold in Stage-specific Embryonic Glycotypes Uncovered by a Full Portrait of Dynamic N-Glycan Expression during Cell Differentiation

Maho Amano; Misa Yamaguchi; Yasuhiro Takegawa; Tadashi Yamashita; Michiyo Terashima; Jun-ichi Furukawa; Yoshiaki Miura; Yasuro Shinohara; Norimasa Iwasaki; Akio Minami; Shin-Ichiro Nishimura

Although various glycoforms appear to participate independently in multiple molecular interactions in cellular adhesion that contribute to embryogenesis and organogenesis, a full portrait of the glycome diversity and the effect of the structural variations of cellular glycoforms on individual cell stages in proliferation and differentiation remain unclear. Here we describe a novel concept for the characterization of dynamic glycoform alteration during cell differentiation by means of “glycoblotting-based cellular glycomics,” the only method allowing for rapid and quantitative glycan analysis. We demonstrated that processes of dynamic cellular differentiation of mouse embryonic carcinoma cells, P19CL6 and P19C6, and mouse embryonic stem cells into cardiomyocytes or neural cells can be monitored and characterized quantitatively by profiling entire N-glycan structures of total cell glycoproteins. Whole N-glycans enriched and identified by the glycoblotting method (67 glycans for P19CL6, 75 glycans for P19C6, and 72 glycans for embryonic stem cells) were profiled and bar-coded quantitatively with respect to the ratio of subgroups composed of characteristic glycoforms, namely glycotypes.


PLOS ONE | 2013

Serum Glycan Markers for Evaluation of Disease Activity and Prediction of Clinical Course in Patients with Ulcerative Colitis

Koji Miyahara; Kazuhiro Nouso; Shunsuke Saito; Sakiko Hiraoka; Keita Harada; Sakuma Takahashi; Yuki Morimoto; Sayo Kobayashi; Fusao Ikeda; Yasuhiro Miyake; Hidenori Shiraha; Akinobu Takaki; Hiroyuki Okada; Maho Amano; Kazuko Hirose; Shin-Ichiro Nishimura; Kazuhide Yamamoto

Background The aims of this study were to determine the change of whole-serum N-glycan profile in ulcerative colitis (UC) patients and to investigate its clinical utility. Methods We collected serum from 75 UC patients at the time of admission and the same number of age/sex-matched healthy volunteers. Serum glycan profile was measured by comprehensive quantitative high-throughput glycome analysis and was compared with disease activity and prognosis. Results Out of 61 glycans detected, 24 were differentially expressed in UC patients. Pathway analysis demonstrated that highly sialylated multi-branched glycans and agalactosyl bi-antennary glycans were elevated in UC patients; in addition, the glycan ratio m/z 2378/1914, which also increased in UC, showed the highest Area under Receiver Operating Characteristic curve (0.923) for the diagnosis of UC. Highly sialylated multi-branched glycans and the glycan ratio m/z 2378/1914 were higher in the patients with total colitis, Clinical Activity Index >10, Mayo endoscopic score 3, or a steroid-refractory status. In particular, the glycan ratio m/z 2378/1914 (above median) was an independent prognostic factor for the need for an operation (hazard ratio, 2.67; 95% confidence interval, 1.04–7.84). Conclusions Whole-serum glycan profiles revealed that the glycan ratio m/z 2378/1914 and highly sialylated multi-branched glycans increase in UC patients, and are correlated with disease activity. The glycan ratio m/z 2378/1914 was an independent predictive factor of the prognosis of UC.


FEBS Journal | 2011

Alteration of the N‐glycome of bovine milk glycoproteins during early lactation

Shota Takimori; Hideyuki Shimaoka; Jun Ichi Furukawa; Tadashi Yamashita; Maho Amano; Naoki Fujitani; Yasuhiro Takegawa; Lennart Hammarström; Imre Kacskovics; Yasuro Shinohara; Shin-Ichiro Nishimura

Milk provides nutritional, immunological and developmental components for newborns. Whereas identification of such components has been performed by targeting proteins and free oligosaccharides, structural and functional analyses of the N‐glycome of milk glycoproteins are scarce. In this study, we investigated, for the first time, the alterations of the bovine milk N‐glycome during early lactation (1 day, 1, 2, 3 and 4 weeks postpartum), characterizing more than 80 N‐glycans. The glycomic profile of colostrum on day 1 after calving differed substantially from that in other periods during early lactation. The proteins in colostrum obtained 1 day postpartum were more highly sialylated than milk samples obtained at other time points, and the N‐glycolylneuraminic acid (Neu5Gc)/N‐acetylneuraminic acid (Neu5Ac) ratio was significantly higher on day 1, showing a gradual decline with time. In order to dissect the N‐glycome of colostrum, alterations of the N‐glycosylation profile of major bovine milk proteins during the early lactation stage were elucidated, revealing that the alteration is largely attributable to qualitative and quantitative N‐glycosylation changes of IgG, the major glycoprotein in colostrum. Furthermore, by preparing and analyzing IgGs in which the N‐glycan structure and subtypes were well characterized, we found that the interaction between IgG and FcRn was not affected by the structure of the N‐glycans attached to IgG. We also found that bovine FcRn binds IgG2 better than IgG1, strongly suggesting that the role of FcRn in the bovine mammary gland is to recycle IgG2 from the udder to blood, rather than to secrete IgG1 into colostrum.


The Journal of Urology | 2014

Serum N-glycan alteration associated with renal cell carcinoma detected by high throughput glycan analysis.

Shingo Hatakeyama; Maho Amano; Yuki Tobisawa; Tohru Yoneyama; Norihiko Tsuchiya; Tomonori Habuchi; Shin-Ichiro Nishimura; Chikara Ohyama

PURPOSE Biomarkers for the early detection and prediction of survival in patients with renal cell carcinoma have not been established. We developed what is to our knowledge a novel glycoblotting method that allows high throughput, comprehensive, quantitative analysis of glycans in human serum. In this study we identified alterations in serum N-glycans associated with renal cell carcinoma. MATERIALS AND METHODS We performed a comprehensive N-glycan structural analysis of serum from 64 patients with renal cell carcinoma and 34 age matched, healthy volunteers using glycoblotting methods and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The peak intensity of N-glycan was analyzed using logistic regression analysis and ROCs were used to select candidate N-glycans. Candidate N-glycans with a statistically significant relationship to renal cell carcinoma or overall survival were independently evaluated using a Cox regression model to determine superiority compared to other conventional renal cell carcinoma biomarkers. RESULTS We identified 56 types of N-glycans in serum from healthy volunteers and patients with renal cell carcinoma. Peaks 40 and 43 were significantly more intense in patients than in volunteers. Peak 19 intensity was significantly higher and peak 49 intensity was significantly lower in patients with renal cell carcinoma who survived for a longer period. Multivariate analysis revealed that peaks 19 and 49 were independent predictors of overall survival. CONCLUSIONS Serum N-glycan analysis is a promising approach to discovering new biomarkers for renal cell carcinoma. Further study is warranted to validate our results.


Hepatology | 2014

Serum glycan as a prognostic marker in patients with advanced hepatocellular carcinoma treated with sorafenib

Koji Miyahara; Kazuhiro Nouso; Yasuhiro Miyake; Shinichiro Nakamura; Shuntaro Obi; Maho Amano; Kazuko Hirose; Shin-Ichiro Nishimura; Kazuhide Yamamoto

els did not see any fibrosis or unphysiological subcapsular fibrosis just after intraperitoneal virus injection. Other researchers have also described the need for strong “danger signals” to break tolerance against liver antigens; however, we could show that the mere overexpression of the autoantigen is not sufficient to cause AIH. This is well in line with the experience of Lapierre et al. employing a repeated prime boost protocol with DNA vaccination with a plasmid encoding for a secreted chimeric antigen of cytochrome P4502D6 (CYP2D6) and formiminotransferase cyclodeaminase (FTCD), with the addition interleukin (IL)-12 or expression with an adenovirus. Of note, we have also tested intramuscular DNA injection with cytomegalovirus-promoter-driven FTCD and IL-12, but did not see meaningful AIH within 6 months in NOD mice (see Fig. 1). The important publication by Bowen et al. did not use danger signals, but rather induced a self-limited hepatitis, followed by immune tolerance, thereby, rather supporting our statement. Regarding the nature of the inducing autoantigen, we could show that similar antigens can induce AIH as well as identical antigens. However, we did not try CYP2D6 as an orthologous autoantigen. The amount of virus could be excluded as a variable, because we tried 1 3 10 to 1 3 10 plaque-forming units of our adenovirus, as reported by Holdener et al. As recently discussed, we think that all the mentioned models are important and can be used to explain various aspects of AIH and hepatic immune tolerance.


Angewandte Chemie | 2012

Glycomics for Drug Discovery: Metabolic Perturbation in Androgen-Independent Prostate Cancer Cells Induced by Unnatural Hexosamine Mimics†

Shin-Ichiro Nishimura; Megumi Hato; Satoshi Hyugaji; Fei Feng; Maho Amano

Inhibited: N-acetylglucosamine (GlcNAc) derivatives with a fluorine atom at the C4 position (2-4) were synthesized, and their ability to inhibit cancer-cell growth was investigated. The administration of these 4F-GlcNAc derivatives to cells led to the unnatural sugar nucleotide 1. Furthermore, N-glycan profiles of cells were determined by using a glycoblotting-based enrichment analysis, which is suitable for high-throughput screenings for drug discovery.


Biochimica et Biophysica Acta | 2014

A straightforward protocol for the preparation of high performance microarray displaying synthetic MUC1 glycopeptides

Takahiko Matsushita; Wataru Takada; Kota Igarashi; Kentaro Naruchi; Risho Miyoshi; Fayna Garcia-Martin; Maho Amano; Hiroshi Hinou; Shin-Ichiro Nishimura

BACKGROUND Human serum MUC1 peptide fragments bearing aberrant O-glycans are secreted from columnar epithelial cell surfaces and known as clinically important serum biomarkers for the epithelial carcinoma when a specific monoclonal antibody can probe disease-relevant epitopes. Despite the growing importance of MUC1 glycopeptides as biomarkers, the precise epitopes of most anti-MUC1 monoclonal antibodies remains unclear. METHODS A novel protocol for the fabrication of versatile microarray displaying peptide/glycopeptide library was investigated for the construction of highly sensitive and accurate epitope mapping assay of various anti-MUC1 antibodies. RESULTS Selective imine-coupling between aminooxy-functionalized methacrylic copolymer with phosphorylcholine unit and synthetic MUC1 glycopeptides-capped by a ketone linker at N-terminus provided a facile and seamless protocol for the preparation of glycopeptides microarray platform. It was demonstrated that anti-KL-6 monoclonal antibody shows an extremely specific and strong binding affinity toward MUC1 fragments carrying sialyl T antigen (Neu5Acα2,3Galβ1,3GalNAcα1→) at Pro-Asp-Thr-Arg motif when compared with other seven anti-MUC1 monoclonal antibodies such as VU-3D1, VU-12E1, VU-11E2, Ma552, VU-3C6, SM3, and DF3. The present microarray also uncovered the occurrence of IgG autoantibodies in healthy human sera that bind specifically with sialyl T antigen attached at five potential O-glycosylation sites of MUC1 tandem repeats. CONCLUSION We established a straightforward strategy toward the standardized microarray platform allowing highly sensitive and accurate epitope mapping analysis by reducing the background noise due to nonspecific protein adsorption. GENERAL SIGNIFICANCE The present approach would greatly accelerate the discovery research of new class autoantibodies as well as the development of therapeutic mAbs reacting specifically with disease-relevant epitopes.

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