Strategy for Glycoproteomics: Identification of Glyco-Alteration Using Multiple Glycan Profiling Tools

Ito, Hiromi; Kuno, Atsushi; Sawaki, Hiromichi; Sogabe, Masamichi; Ozaki, Haruka; Tanaka, Yasuhito; Mizokami, Masashi; Shoda, Junichi; Angata, Takashi; Sato, Takashi; Hirabayashi, Jun; Ikehara, Yuzuru; Narimatsu, Hisashi

doi:10.1021/pr800735j

Cited by 67 publications

(87 citation statements)

References 47 publications

(87 reference statements)

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“…With special focus on cancer-associated glyco-alteration, we recently proposed a robust strategy to develop high performance glycoprotein biomarkers using advanced technologies of glycopropteomics. 17,34 Along with the established strategy, we attempted differential glycan analysis using tissue sections containing both normal BDE and ICC lesions from the same patients. An ultrasensitive glycan profiling technology, lectin microarray mined WFA 30 as the most promising probe to differentiate ICC from normal BDE with a significant P value (<0.0001).…”

Section: Discussionmentioning

confidence: 99%

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Wisteria floribunda agglutinin-positive mucin 1 is a sensitive biliary marker for human cholangiocarcinoma

Matsuda

Kuno

Kawamoto³

et al. 2010

Hepatology

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Cholangiocarcinoma (CC) is an aggressive malignant tumor for which useful markers are not presently available for early and precise diagnosis. The aim of this study was therefore to identify a high-performance diagnostic marker with a special focus on glyco-alteration of glycoproteins. In the course of study, we found that Wisteria floribunda agglutinin (WFA) is the best probe to differentiate intrahepatic cholangiocarcinoma (ICC) lesions from normal bile duct epithelia (BDE) (P < 0.0001). The subsequent histochemical study confirmed ICC-specific WFA staining on 165 tissue specimens. On the other hand, the WFA staining was shown to be closely associated with that of MY.1E12 established previously against sialylated mucin 1 (MUC1) by double-staining experiments. Moreover, glyco-alteration of MUC1 could be verified by western blotting of WFA-captured bile samples from patients with CC patients. Thus, we attempted to construct an enzyme-linked immunosorbent assay system for more convenient CC diagnosis, where WFA-coated plates, the specific monoclonal antibody MY.1E12, and the bile specimens from CC including ICC (n 5 30) and benign diseases (n 5 38) were combined. As a result, CC was clearly distinguished from benign diseases with statistical scores (sensitivity 5 90.0%, specificity 5 76.3%, and area under the curve 5 0.85). As a particular note, the obtained sensitivity is the highest score among those having been so far reported. Conclusion: Our approach focusing significant glyco-alteration of a particular glycoprotein yielded a novel diagnostic system for CC with satisfactory clinical scores. (HEPATOLOGY 2010;52:174-182) C holangiocarcinoma (CC) is an aggressive malignant tumor arising from the epithelial lining of the intrahepatic biliary tract. Although it contributes to only 15% of the total incidence of primary liver cancer, 1 recent epidemiological reports show that the CC incidence has increased significantly in Abbreviations: AFP, alpha-fetoprotein; AUC, area under the curve; BDE, bile duct epithelia; BSA, bovine serum albumin; CA19-9, carbohydrate antigen 19-9;

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Section: Discussionmentioning

confidence: 99%

“…This difference in expression emphasizes the importance of glycosylation change (glyco-alteration) occurring on the same protein. 17 Thus, glyco-alteration of particular glycoproteins has attracted increasing attention among biomarker investigators.…”

mentioning

confidence: 99%

Wisteria floribunda agglutinin-positive mucin 1 is a sensitive biliary marker for human cholangiocarcinoma

Matsuda

Kuno

Kawamoto³

et al. 2010

Hepatology

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“…Multiple q-PCR was performed as previously described. (30) Briefly, cDNA was synthesized from 4 lg of total RNA, and then subjected to a q-PCR array with 186 glycogenes and three housekeeping genes. Copy numbers of transcripts in 7.5 ng of total RNA were calculated, and those under 100 were eliminated from the evaluation.…”

Section: Methodsmentioning

confidence: 99%

Membrane sialidase NEU3 is highly expressed in human melanoma cells promoting cell growth with minimal changes in the composition of gangliosides

Miyata¹,

Kambe²,

Tajima³

et al. 2011

Cancer Science

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NEU3 is a membrane sialidase specific for gangliosides. Its increased expression and implication in some cancers have been reported. Here, we analyzed NEU3 expression in malignant melanoma cell lines and its roles in the cancer phenotypes. Quantitative RT-PCR revealed that high levels of the NEU3 gene were expressed at almost equivalent levels with those in colon cancers. To examine the effects of overexpression of NEU3, NEU3 cDNA-transfectant cells were established using a melanoma cell line SK-MEL-28 and its mutant N1 lacking GD3. SK-MEL-28 sublines overexpressing both the NEU3 gene and NEU3 enzyme activity showed no changes in both cell growth and ganglioside expression, while N1 cells showed a mild increase in cell proliferation with increased phosphorylation of the EGF receptor and neo-synthesis of Gb3 after NEU3 transfection. In contrast, NEU3 silencing resulted in a definite reduction in cell growth in a melanoma line MeWo, while ganglioside patterns underwent minimal changes. Phosphorylation levels of ERK1 ⁄ 2 with serum stimulation decreased in the NEU3-silenced cells. All these results suggest that NEU3 is highly expressed to enhance malignant phenotypes including apoptosis inhibition in malignant melanomas. (Cancer Sci 2011; 102: 2139-2149

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“…12,13) These genes have been cloned into the entry vector pENTR D TOPO (Invitrogen Japan KK., Tokyo, Japan). Certain glycosyltransferases encode the enzymes involved in the synthesis of lipid linked oligosaccharide intermediates in the lumen side of the endoplasmic reticulum (ER).…”

Section: Expression Of Glycosyltransferases In Hek293t Cellsmentioning

confidence: 99%

Expression System for Human Glycosyltransferases and Its Application

Chiba¹,

Ito²,

Sato³

et al. 2010

J. Appl. Glycosci.

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Glycosylation of proteins and lipids is the most complex of their various co translational and post translational modifications. Glycan modification contributes to the folding and conformational stability of many proteins, 1) and modified glycoproteins and glycolipids on the cell surface are known to act as ligands for host pathogen interactions, 2) cell adhesion and cell signaling.3) These phenomena are influenced by changes in glycan structures. Since it is known that glycans on proteins and lipids are synthesized by sequential reactions by glycosyltransferases, the alteration of the glycan structure is dependent on the expression of glycan synthesis related genes encoding glycosyltransferases, sulfotransferases, glycosidases, sugar nucleotide synthetases, and sugar nucleotide transporters. Glycan structures are defined by not only the expression level of the genes but also several other factors, such as substrate specificity, competition of more than two glycosyltransferases for the same substrate, localization of glycosyltransferases, and concentration of donor substrate as sugar nucleotides in the Golgi apparatus. Therefore, the expression of glycosyltransferases and the determination of their enzymatic characterizations in detail are significant for the elucidation of the biosynthetic machinery of glycans.In addition to basic study of glycosyltransferases, recombinant enzymes are available for the synthesis of glycans and glycopeptides and for the modification of glycoproteins in vitro. In recent years, mass spectrometry, 4,5) high performance liquid chromatography (HPLC), 6,7) and lectin microarray systems 8,9) have been developed for the structural analysis of glycans. A glycan library composed of a variety of structure defined glycans is indispensable for quantitative analyses using these technologies. Since human glycosyltransferases have a strict specificity toward donor and acceptor substrates, it is reasonable to employ in vitro synthesis of glycans by glycosyltransferases for producing glycan standards. Glycan array technology, and the ease with which the glycans can be customized for display, may enhance our ability to detect unknown lectins in mammals and to probe pathogen specific glycan interactions. 10,11) Application of glycosyltransferases in other areas of interest includes the modification of glycans attached to glycoproteins, which has also become an important topic in recent years. Because biologics and biosimilar drugs such as therapeutic antibodies and cytokines are the largest class of new drug candidates being developed by pharmaceutical companies, there are strong concerns that the heterogeneity of the glycan structure may sometimes affect their in vivo activity. Therefore, the development of technology to produce glycoproteins with homogeneous glycans is essential.In our research center, the Glycogene (GG) Project was started in 2001, in which as many novel genes as possible, which were the candidates for glycogenes including glycosyltransferases and sugar nucleotide transporters, ...

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Strategy for Glycoproteomics: Identification of Glyco-Alteration Using Multiple Glycan Profiling Tools

Cited by 67 publications

References 47 publications

Wisteria floribunda agglutinin-positive mucin 1 is a sensitive biliary marker for human cholangiocarcinoma

Wisteria floribunda agglutinin-positive mucin 1 is a sensitive biliary marker for human cholangiocarcinoma

Membrane sialidase NEU3 is highly expressed in human melanoma cells promoting cell growth with minimal changes in the composition of gangliosides

Expression System for Human Glycosyltransferases and Its Application

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