Development of Advanced Technology and Its Application to Discovery of Cancer Glyco-biomarker

Hisashi Narimatsu 한국당과학회 2008 한국당과학회 학술대회 Vol.2008 No.1

For these 7 years, we have been developing new technologies for Glycomics with the sponsorship of the New Energy and Industrial Technology Development Organization (NEDO). We discovered new glyco-genes and constructed human glyco-gene library consisting of 184 genes. Knowledge of the substrate specificities of these gene products allowed us to better understand the molecular basis of human glycosylation. Taking full advantage of our glyco-gene library, we developed a glycan library that was then used as standards to develop instruments for glycan structural analysis, such as mass spectrometer-based glycan sequencer and lectin microarray-based glycan profiler. Association of aberrant glycosylation with cancer has long been recognized. However, much of the discovery still depends on serendipity, and biological meanings of these cancer-related glycosylation patterns are only gradually uncovered. In 2006, we launched a new project termed Medical Glycomics (MG) project. Our aims in the project are two-folds: (1) development of discovery systems for cancer-related glyco -biomarkers, and (2) functional analysis of the cancer-associated glycosylation. We present many candidates for cancer-glyco-biomarker which would be useful for diagnosis.

A chemoenzymatic approach toward the identification of fucosylated glycoproteins and mapping of N-glycan sites

Ta-Wei Liu,Hiroyuki Kaji,Akira Togayachi,Hiromi Ito,Kiyohiko Angata,Takashi Sato,Hisashi Narimatsu 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

Fucose-containing glycoconjugates play important roles in numerous physiological and pathological processes. Given the biological importance of posttranslational glycosylation, a specific and robust strategy for the identification of fucosylated glycoproteins is highly desirable. In this study, we demonstrate an alternative way of labeling of fucosylated structures by metabolic engineering, using a chemoenzymatic approach. In this approach, the activities of Bacteroides fragilis 9343 L-fucokinase/GDP-fucose pyrophosphorylase and human α1,3-fucosyltransferase 9 are combined in a Namalwa cellular model. Interestingly, this system could be applied to labeling of alkyne-modified fucosylated glycoproteins. N-glycan site mapping and identification was done using an in vitro selective chemical ligation reaction and isotope-coded glycosylation site-specific tagging, subsequent to liquid chromatography-tandem mass spectrometry analysis. This work illustrates the use of a click chemistry-based strategy combined with a glycoproteomic technique to get further insight into the pattern of fucose-mediated biological processes and functions.

Overview of JCGGDB including New Released GlycoProtDB

Toshihide Shikanai,Hiroyuki Kaji,Yoshinori Suzuki,Noriaki Fujita,Masako Maeda,HonglingWen,Madoka Ishizaki,Hiromichi Sawaki,Hisashi Narimatsu. 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

The JST/NBDC integrated database project has kicked off last year. JCGGDB was selected as a promotion program of DB integration, aiming to integrate all the glycan-related databases in Japan and build user- friendly search systems. As part of the project, the construction of ACGG-DB (an integrated database for the ACGG: Asian Communications for Glycobiology and Glycotechnology) is also planned in cooperation with Asian countries. As of now we have consolidated data from various Japanese institutes into JCGGDB and developed a cross-search function by keyword entry and integrated search functions by glycan stcurctures. These functions enabled users to easily access various glycan-related databases with a single search. Cheminformatics technologies using chemical structural formula for glycan has been also adopted to provide a search for glycan structures, glycan synthetic products by organic chemistry and recombinant enzymes, glycogene inhibitors, glycosides, and commercial glycans. This Summer, we have released AIST GlycoProtDB, which stores the data of experimentally-proven glycosylation sites on each mouse tissue. We are continuously accumulating experimental results of glycosylation sites, while collecting more information from scientific journals, toward the release of ACGG Glycoprotein Database in autumn. For the future, we will keep developing base technologies for DB integration and linking with databases related to glycoscience as well as other study areas. Some more bioinformatics tools are also being developed to support experimental study. Our aim is to create contents which could be easily and intuitively understood by every user.

X-ray Crystallographic Studies on Binding Specificity of Norovirus to Lewis Antigens

Tomomi Kubota,Akiko Kumagai,Hiromi Ito,Sanae Furukawa,Yuichi Someya,Koji Ishii,Takaji Wakita,Naokazu Takeda,Haruko Shirato,Hisashi Narimatsu 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

Norovirus (NoV) is a major causative agent of nonbacterial acute, epidemic gastroenteritis, and often widespread in winter. Although most NoV infections are cured without serious symptoms, it could cause severe symptoms in elderlies and young children. The initial infection event is adhesion of NoV to glycans expressed in the epidermal cells of the small intestine. We have identified several NoV strains that show affinity to Le

Glycomics meets histopathology: Lectin microarray can profile the whole glycoproteins in specific groups of cells isolated by laser microdissection

Atsushi Kuno,Masaharu Nomura,Hideki Matsuzaki,Tomoko Nakagawa,Atsushi Matsuda,Yoshitoshi Hirao,Masao Sasaki,Norihiro Ikeda,Toshitaka Nagao,Yuzuru Ikehara,Hisashi Narimatsu 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

Cell glycome is defined by the glyco synthesis machinery regulated by harmonized expression of more than 100 glycogenes. The machinery-dependent glycome drastically shifts during cell progression and differentiation in association with tumorigenesis and malformation, and thus it motivates us to discover the disease-related alteration in glycosylation. Glycan-targeted histochemical approaches using lectin and anti-glycogene antibodies have provided some key information to characterize specific histological types of cells in pathology. However, this approach is not suitable for the comprehensive analysis targeting the cell glycome, and thus may fail to provide insight into glycome shift during the disease progression. Several years ago, we developed the methodology for rapid and systematic glycome shift analysis targeting formalin-fixed tissue specimens by means of lectin microarray. The resultant method enabled simultaneous observation of over 40 lectins interacted with glycoproteins in 1 mm2 of the tissue specimens. Recently, we sophisticated this methodology to be suitable for comparative analysis of a series of cells in specific groups isolated from a single tissue specimen by laser microdissection, and now our research has gained interest in the variability and distribution of cell glycome in the tissue, i.e., “tissue glycome mapping”. In this meeting, we will summarize the advantage of this new methodology and its application for glyco-biomarker discovery, as well as the construction of “tissue glycome atlas”.

Identification of glyco-biomarker candidates for lung cancer using novel glyco-technologies

Yoshitoshi Hirao,Hideki Matsuzaki,Jun Iwaki,Minako Abe,Akira Togayachi,Atsushi Kuno,Takashi Ohkura,Hiroyuki Kaji,Masaharu Nomura,Masayuki Noguchi,Yuzuru Ikehara,Hisashi Narimatsu 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

Lung cancer is the leading cause of cancer death worldwide. Currently, lung cancer is classified into two major types, small-cell lung cancer carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC), based on the histological appearance. The histological classification has important implications in the clinical practice guideline and the prediction of the patient prognosis. However, conventional serum markers used in clinical tests are insufficient for clinical demands due to the low sensitivity and the low specificity to distinguish them. We have identified a number of glyco-biomarker candidate molecules from lung cancer cell lines using our developed glycoproteomics technologies such as lectin microarray and LC/MS-based protein analysis. On the validation studies, we found out that the selected molecules showed characteristic lectin biding profiles depending on either SCLC or NSCLC. Therefore, combination of these glyco-biomarkers could be expected to improve the diagnostic accuracy for histological classification in lung cancer compared to protein expression alone.

Verification of Wisteria floribunda agglutinin-positive glycoproteins as a cholangiocarcinoma marker

Atsushi Matsuda,Atsushi Kuno,Hideki Matsuzaki,Toru Kawamoto,Toshihide Shikanai,Yasuni Nakanuma,Masakazu Yamamoto,Nobuhiro Ohkohchi,Yuzuru Ikehara,Junichi Shoda,Jun Hirabayashi,Hisashi Narimatsu 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

Cholangiocarcinoma (CC) is a lethal malignancy which exhibits asymptomatic growth infiltrating the surrounding structures, and thus,CC is usually detected at an advanced stage. The mainstay of treatment for CC is complete resection with negative surgical margins. Therefore, its diagnosis at a relatively early stage is demanded for performing the surgical resection. Since the definitive CC diagnosis relies on invasive methods such as biliary cytology and biopsy, a noninvasive assay with high diagnostic accuracy is keenly required. In the previous meeting, we reported that Wisteria floribunda agglutinin (WFA) is the best probe lectin which reliably distinguishes between CC and normal bile duct epithelia in tissue sections. Moreover, L1 cell adhesion molecule (L1CAM), CA125, and maspin were assigned as the reliable CC marker candidates by WFA-assisted glycoproteomics and immunohistochemistry. In this meeting, we will introducethe verification and validation process in one of the above candidates, L1CAM. Since the serum concentration of L1CAM was low as described in other reports (< 5 ng/mL), we firstly constructed a highly-sensitive detection system to confirm the existence of L1CAM in both bile and serum of CC patients with immunoprecipitation and western blotting. We then performed highly-sensitive glycan profiling with antibody-assisted lectin microarray (limit of detection: 25 pg) and confirmed WFA-positivity of biliary L1CAM from the CC patients. The subsequent validation study using bile samples from CC patients (n = 29) and patients with benign bile duct diseases (n = 29) showed that WFA-positive L1CAM distinguished CC from the benigndiseases with good specificity (sensitivity = 0.66, specificity = 0.93, overall accuracy = 0.79, area under the receiver operating curve [AUC] = 0.82). The combined use of the L1CAM assay with the highly-sensitive assay detecting WFA-positive sialylated mucin 1 (WFA-sialyl MUC1), a reliable CC marker (Matsuda A., et al., Hepatology, 2010), sufficiently improved the diagnostic accuracy of CC (overall accuracy = 0.84, AUC = 0.93). This combination assay using WFA–L1CAM and WFA–sialyl MUC1 will possibly be a useful serological test with enhanced reliability.