Kazuko Hirose

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

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.

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

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.

Insight into glycan diversity and evolutionary lineage based on comparative Avio-N-glycomics and sialic acid analysis of 88 egg whites of Galloanserae.

A large set of glycome information was obtained from egg white proteins of 88 samples from Galloanserae (63 Anseriformes and 25 Galliformes). The data were obtained on whole N-glycan structures and types of sialic acids of these egg whites by glycoblotting-based high-throughput and quantitative glycomics. The results revealed clear trends and complexity patterns as well as diversity among taxonomic groups. It is well-known that chicken, a representative domesticated poultry involved in Galliformes, can become an influenza host. However, our data demonstrate that duck, wild goose, and swan of Anseriformes are representative migratory birds that are known as natural hosts of the influenza virus. Hierarchical clustering analysis of the expression pattern of N-glycome (total of 61 N-glycan peaks) revealed that the members of Galloanserae can be classified into two major groups and five submajor clusters (clusters 1-5) on the basis of simple m/z values obtained by MALDI-TOF MS. It is clear that expression patterns of N-glycomes in the five clusters are influenced significantly by the features such as the body size of the birds, rather than by the difference of the family. On the other hand, quantitative analysis showed that the total amounts of sialic acids in egg whites of Galliformes were distinctly larger than those of Anseriformes. However, it was also revealed in Anseriformes that Neu5Gc and KDN, in addition to common Neu5Ac, were expressed significantly in both N- and O-glycans of glycoproteins and glycosphingolipids, suggesting the influence of their lifestyles and diet. This is the first report that KDN exists in egg white. These results and the environmental factors are discussed preliminarily with respect to their evolutionary lineage.

N- and O-glycome analysis of serum and urine from bladder cancerpatients using a high-throughput glycoblotting method

Purpose: To develop novel diagnostic biomarkers for Bladder Cancer (BC), we concurrently evaluated serum and urine glycans in BC patients by utilizing a recently established glycoblotting method. Experiments: N- and O-glycan levels in whole serum from 45 and 13 male patients diagnosed with BC were analyzed. As a control, 29 and 10 patients with benign prostatic hyperplasia (BPH) were also studied and the results were compared. Furthermore, urine N- and O-glycome from 8 patients with muscle-invasive BC and 11 with BPH were analyzed. In serum N-glycome analysis the area-under-the-curve (AUC) value was calculated by in house R software. In the other cases JMP, version 10.0.2 software package (SAS, Cary, NC) was used and p <0.05 was considered statistically significant. Results: The expression level of three N-glycans significantly increased in sera of BC patients. All of them had highly branched and sialylated structures with core-Fuc. The levels of three O-glycans were significantly higher in BC than in the control. In examination of urine samples, 16 N-glycans were significantly elevated in BC as compared to the control. Although 11 O-glycans were detected in urine samples, there was no significant difference in the expression levels. Conclusions: The levels of highly branched, sialylated N-glycans and early sialylated O-glycans were increased in sera of BC patients. Moreover, we found that N-glycans increased in urine more than in serum of BC patients. These results suggest that the glycome change in urine directly results from glycoproteins that exist in BC cells. Thus, further large-scale glycan profiling will provide novel biomarkers for diagnosing BC in the near future.

N-Glycomic and Microscopic Subcellular Localization Analyses of NPP1, 2 and 6 Strongly Indicate that trans-Golgi Compartments Participate in the Golgi to Plastid Traffic of Nucleotide Pyrophosphatase/Phosphodiesterases in Rice

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1–NPP6. Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)–Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2–GFP and NPP6–GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER–Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs.

Functional network of glycan-related molecules: Glyco-Net in Glycoconjugate Data Bank

BackgroundGlycans are involved in a wide range of biological process, and they play an essential role in functions such as cell differentiation, cell adhesion, pathogen-host recognition, toxin-receptor interactions, signal transduction, cancer metastasis, and immune responses. Elucidating pathways related to post-translational modifications (PTMs) such as glycosylation are of growing importance in post-genome science and technology. Graphical networks describing the relationships among glycan-related molecules, including genes, proteins, lipids and various biological events are considered extremely valuable and convenient tools for the systematic investigation of PTMs. However, there is no database which dynamically draws functional networks related to glycans.DescriptionWe have created a database called Glyco-Net http://www.glycoconjugate.jp/functions/, with many binary relationships among glycan-related molecules. Using search results, we can dynamically draw figures of the functional relationships among these components with nodes and arrows. A certain molecule or event corresponds to a node in the network figures, and the relationship between the molecule and the event are indicated by arrows. Since all components are treated equally, an arrow is also a node.ConclusionsIn this paper, we describe our new database, Glyco-Net, which is the first database to dynamically show networks of the functional profiles of glycan related molecules. The graphical networks will assist in the understanding of the role of the PTMs. In addition, since various kinds of bio-objects such as genes, proteins, and inhibitors are equally treated in Glyco-Net, we can obtain a large amount of information on the PTMs.

Functional Network in Posttranslational Modifications: Glyco-Net in Glycoconjugate Data Bank

Elucidating pathways related to posttranslational modifications (PTMs) such as glycosylation is of growing importance in post-genome science and technology. Graphical networks describing the relationships among glycan-related molecules, including genes, proteins, lipids, and various biological events, are considered extremely valuable and convenient tools for the systematic investigation of PTMs. Glyco-Net (http://bibi.sci.hokudai.ac.jp/functions/) can dynamically make network figures among various biological molecules and biological events. A certain molecule or event is expressed with a node, and the relationship between the molecule and the event is indicated by arrows in the network figures. In this chapter, we mention the features and current status of the Glyco-Net and a simple example of the search with the Glyco-Net.