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Survey of glycoantigens in cells from α1-3galactosyltransferase knockout pig using a lectin microarray

XENOTRANSPLANTATION 2010年第1期17卷 61-70页

作者： Miyagawa, Shuji Takeishi, Shunsaku Yamamoto, Aki Ikeda, Kosuke Matsunari, Hitomi Yamada, Masao Okabe, Masaru Miyoshi, Eiji Fukuzawa, Masahiro Nagashima, Hiroshi Osaka Univ Grad Sch Med Dept Surg Div Organ Transplantat E9 Suita Osaka 5650871 Japan Moritex Corp Glyc Res Lab Yokohama Kamagra Japan Meiji Univ Dept Life Sci Kawasaki Kamagra Japan Osaka Univ Microbial Dis Res Inst Osaka Japan Osaka Univ Grad Sch Med Dept Mol Biochem & Clin Invest Osaka Japan

Background: Glycoantigens represent major obstacles to successful xenotransplantation. Even after the alpha 1-3galactosyltransferase (GalT) gene knockout (GalT-KO) pigs were produced, non-Gal antigens continue to be present. This study reports on lectin blot analyses for endothelial cells (EC) and fibroblasts from GalT-KO pigs. Methods: Differences in glycoantigens that are produced on cell surfaces in humans and pigs were surveyed. Differences between ECs and fibroblasts from wild-type and GalT-KO pigs were also examined. EC and fibroblasts from GalT-KO pigs (heterozygous and homozygous) with N-acetylglucosaminyltransferase-III (GnT-III), a wild-type EC from the sibling, human EC lines, HUVEC (human EC from umbilical veins), & HAOEC (human EC from aortas), and human fibroblast line were used. EC and fibroblasts were cultured in gelatin-coated dishes for several days. After sonication and centrifugation, the supernatant protein from each cell was labeled with Cy3, applied to a lectin array and scanned with an SC Profiler, and analyzed using an Array Pro Analyzer. Results: The pig EC showed higher signals in Euonymus Europaeus (EEL) & Griffonia simplicifolia I-B-4 (GSI-B4), binds alpha-Gal, and in Wisteria Floribunda (WFA), Helix pomatia (HPA), Glycine max (SBA), & Griffonia simplicifolia I-A(4) (GSI-A4), binds GalNAc including the Thomsen-Friedenreich precursor (Tn)-antigen, while the human EC showed strong signals in Ulex europaeus I (UEA-I), Maackia amurensis (MAL), Erythrina cristagalli (ECA), & Trichosanthes japonica I (TJA-I) instead. The EC from the GalT-KO pig signals for EEL & GSI-B4 disappeared and those for Bauhinia purpurea alba (BPL), HPA, SBA, & GSI-A4 were greatly diminished as well, while it up-regulated signals for Sambucus Nigra (SNA), Sambucus sieboldiana (SSA), & TJA-I, bind alpha 2-6 sialic acid, compared to the wild-type pig EC. Concerning fibroblasts, the signals for HPA, SBA, & GSI-A4 were the most intense in the wild-type, and the intensities

关键词： Gal knockout pig glyco-antigen lectin microarray non-Gal antigen

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Altered glycosylation associated with dedifferentiation of hepatocellular carcinoma: a lectin microarray-based study

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BMC CANCER 2020年第1期20卷 192-192页

作者： Takayama, Hiroomi Ohta, Masayuki Iwashita, Yukio Uchida, Hiroki Shitomi, Yuki Yada, Kazuhiro Inomata, Masafumi Oita Univ Fac Med Dept Gastroenterol & Pediat Surg 1-1 IdaigaokaHasama Machi Oita 8795593 Japan Oita Univ Global Oita Med Adv Res Ctr Hlth Oita Japan

BackgroundAltered glycosylation associated with hepatocellular carcinoma (HCC) is well documented. However, few reports have investigated the association between dedifferentiation and glycosylation. Therefore, the aim of this study was to analyze glycosylation associated with dedifferentiation of HCC within the same nodule and to investigate glycosyltransferase related to the *** analyzed resected HCC specimens (n=50) using lectin microarray to comprehensively and sensitively analyze glycan profiles, and identify changes to glycosylation between well- and moderately-differentiated components within the same nodule. Moreover, we performed immunohistochemical staining of mannosyl(alpha -1,3-)-glycoprotein beta -1,2-N-acetylglucosaminyltransferase (MGAT1), which is an essential glycosyltransferase that converts high-mannose glycans to complex- or hybrid-type *** lectins from Narcissus pseudonarcissus agglutinin (NPA), Concanavalin A, Galanthus nivalis agglutinin, and Calystegia sepium agglutinin were significantly elevated in moderately-differentiated components of HCC compared with well-differentiated components, and all lectins showed binding specificity to high-mannose glycans. Therefore, these structures were represented to a greater extent in moderately-differentiated components than in well-differentiated ones. Immunohistochemical staining revealed significantly increased NPA expression and decreased MGAT1 expression in moderately-differentiated components. Low MGAT1 expression in moderately-differentiated components of tumors was associated with intrahepatic metastasis and had tendency for poor *** of well-differentiated HCC is associated with an increase in high-mannose glycans. MGAT1 may play a role in the dedifferentiation of HCC.

关键词： Hepatocellular carcinoma Dedifferentiation High-mannose glycan lectin microarray Mannosyl(alpha-1 3-)-glycoprotein beta-1 2-N-acetylglucosaminyltransferas

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Altered N-Glycan Expression Profile in Epithelial-to-Mesenchymal Transition of NMuMG Cells Revealed by an Integrated Strategy Using Mass Spectrometry and Glycogene and lectin microarray Analysis

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JOURNAL OF PROTEOME RESEARCH 2014年第6期13卷 2783-2795页

作者： Tan, Zengqi Lu, Wei Li, Xiang Yang, Ganglong Guo, Jia Yu, Hanjie Li, Zheng Guan, Feng Jiangnan Univ Sch Biotechnol Minist Educ Key Lab Carbohydrate Chem & Biotechnol Wuxi 214122 Jiangsu Peoples R China Jiangnan Univ Wuxi Med Sch Wuxi 214122 Jiangsu Peoples R China NW Univ Xian Coll Life Sci Lab Funct Glyc Xian 710069 Peoples R China

Epithelial-to-mesenchymal transition (EMT) is an essential biological process that occurs in embryonic development, metastatic diseases, and cancer progression. Altered expression of glycans is known to be associated with cancer progression. No studies to date have presented global analysis of the precise variation of N-glycans in EMT. We describe here the profile of N-glycans and glycogene expression in the EMT process induced by transforming growth factor-beta 1 (TGF beta 1) in a normal mouse mammary gland epithelial (NMuMG) cell model. An integrated strategy with a combination of mass spectrometry, glycogene microarray analysis, and lectin microarray analysis was applied, and results were confirmed by lectin histochemistry and quantitative real-time PCR. In TGF beta-induced EMT, levels of high-mannose-type N-glycans were enhanced, antennary N-glycans, and fucosylation were suppressed, and bisecting GIcNAc N-glycans were greatly suppressed. The expression of seven N-glycan-related genes was significantly changed. The products of glycogenes ALG9, MGAT3, and MGAT4B appeared to contribute to the observed alteration of N-glycans. The findings indicate that dysregulation of N-glycan synthesis plays a role in the EMT process. Systematic glycomic analysis based on the combination of techniques described here is expected to facilitate the discovery of the aberrant N-glycosylation in tumor progression and provide essential information in systems glycobiology.

关键词： N-glycan glycogene mass spectrometry gene microarray lectin microarray high throughput

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Identification of Cell Surface Glycoprotein Markers for Glioblastoma-Derived Stem-Like Cells Using a lectin microarray and LC-MS/MS Approach

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JOURNAL OF PROTEOME RESEARCH 2010年第5期9卷 2565-2572页

作者： He, Jintang Liu, Yashu Xie, Xiaolei Zhu, Thant Soules, Mary DiMeco, Francesco Vescovi, Angelo L. Fan, Xing Lubman, David M. Univ Michigan Med Ctr Dept Surg Ann Arbor MI 48109 USA Univ Michigan Med Ctr Dept Neurosurg Ann Arbor MI 48109 USA Univ Michigan Med Ctr Dept Cell & Dev Biol Ann Arbor MI 48109 USA Johns Hopkins Univ Baltimore MD 21205 USA Ist Nazl Neurol Carlo Besta Dept Neurosurg I-20133 Milan Italy Univ Milano Bicocca Dept Biosci & Biotechnol I-20126 Milan Italy

Despite progress in the treatment of glioblastoma, more than 95% of patients suffering from this disease still die within 2 years. Recent findings support the belief that cancer stem-like cells are responsible for tumor formation and ongoing growth. Here a method combining lectin microarray and LC-MS/MS was used to discover the cell surface glycoprotein markers of a glioblastoma-derived stem-like cell line. lectin microarray analysis of cell surface glycans showed that two galactose-specific lectins Trichosanthes kirilowii agglutinin (TKA) and Peanut agglutinin (PNA) could distinguish the stem-like glioblastoma neurosphere culture from a traditional adherent glioblastoma cell line. Agarose-bound TKA and PNA were used to capture the glycoproteins from the two cell cultures, which were analyzed by LC-MS/MS. The glycoproteins were quantified by spectral counting, resulting in the identification of 12 and 11 potential glycoprotein markers from the TKA and PNA captured fractions respectively. Almost all of these proteins were membrane proteins. Differential expression was verified by Western blotting analysis of 6 interesting proteins, including the up-regulated Receptor-type tyrosine-protein phosphatase zeta, Tenascin-C, Chondroitin sulfate proteoglycan NG2, Podocalyxin-like protein 1 and CD90, and the down-regulated CD44. An improved understanding of these proteins may be important for earlier diagnosis and better therapeutic targeting of glioblastoma.

关键词： glycoprotein biomarker lectin microarray glioblastoma stem-like cells LC-MS/MS

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A novel strategy for mammalian cell surface glycome profiling using lectin microarray

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GLYCOBIOLOGY 2007年第10期17卷 1138-1146页

作者： Tateno, Hiroaki Uchiyama, Noboru Kuno, Atsushi Togayachi, Akira Sato, Takashi Narimatsu, Hisashi Hirabayashi, Jun Natl Inst Adv Ind Sci & Technol Res Ctr Med Glycosci Tsukuba 3058568 Japan

The glycome represents the total set of glycans expressed in a cell. The glycome has been assumed to vary between cell types, stages of development and differentiation, and during malignant transformation. Analysis of the glycome provides a basis for understanding the functions of glycans in these cellular processes. Recently, a technique called lectin microarray was developed for rapid profiling of glycosylation, although its use was mainly restricted to glycoproteins of cell lysates, and thus unable to profile the intact cell surface glycans. Here we report a simple and sensitive procedure based on this technology for direct analysis of the live mammalian cell-surface glycome. Fluorescent-labeled live cells were applied in situ to the established lectin microarray consisting of 43 immobilized lectins with distinctive binding specificities. After washing, bound cells were directly detected by an evanescent-field fluorescence scanner in a liquid phase without fixing and permeabilization. The results obtained by differential profiling of CHO and its glycosylation-defective mutant cells, and splenocytes of wildtype and beta 1-3-N-acetylglucosaminyltransferase II knockout mice performed as model experiments agreed well with their glycosylation phenotypes. We also compared cell surface glycans of K562 cells before and after differentiation and found a significant increase in the expression of O-glycans on differentiated cells. These results demonstrate that the technique provides a novel strategy for profiling global changes of the mammalian cell surface glycome.

关键词： glycome lectin microarray live cells profiling

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The use of lectin microarray for assessing glycosylation of therapeutic proteins

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MABS 2016年第3期8卷 524-535页

作者： Zhang, Lei Luo, Shen Zhang, Baolin Food & Drug Adm Ctr Drug Evaluat & Res Off Biotechnol Prod Silver Spring MD USA

Glycans or carbohydrates attached to therapeutic glycoproteins can directly affect product quality, safety and efficacy, and therefore must be adequately analyzed and controlled throughout product life cycles. However, the complexity of protein glycosylation poses a daunting analytical challenge. In this study, we evaluated the utility of a lectin microarray for assessing protein glycans. Using commercial lectin chips, which contain 45 lectins toward distinct glycan structures, we were able to determine the lectin binding patterns of a panel of 15 therapeutic proteins, including 8 monoclonal antibodies. lectin binding signals were analyzed to generate glycan profiles that were generally consistent with the known glycan patterns for these glycoproteins. In particular, the lectin-based microarray was found to be highly sensitive to variations in the terminal carbohydrate structures such as galactose versus sialic acid epitopes. These data suggest that lectin microarray could be used for screening glycan patterns of therapeutic glycoproteins.

关键词： monoclonal antibodies therapeutic glycoproteins lectin microarray Glycan analysis

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High-precision multiclass cell classification by supervised machine learning on lectin microarray data

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REGENERATIVE THERAPY 2020年 15卷 195-201页

作者： Shibata, Mayu Okamura, Kohji Yura, Kei Umezawa, Akihiro Natl Ctr Child Hlth & Dev Dept Reprod Biol Tokyo 1578535 Japan Ochanomizu Univ Grad Sch Humanities & Sci Tokyo 1128610 Japan Natl Ctr Child Hlth & Dev Dept Syst BioMed Tokyo 1578535 Japan Waseda Univ Sch Adv Sci & Engn Tokyo 1620041 Japan

Introduction: Establishment of a cell classification platform for evaluation and selection of human pluripotent stem cells (hPSCs) is of great importance to assure the efficacy and safety of cell-based therapy. In our previous work, we introduced a discriminant function that evaluates pluripotency from the cells' glycome. However, it is not yet suitable for general use. Methods: The current study aims to establish a high-precision cell classification platform introducing supervised machine learning and test the platform on glycome analysis as a proof-of-concept study. We employed linear classification and neural network to the lectin microarray data from 1577 human cells and categorized them into five classes including hPSCs. Results: The linear-classification-based model and the neural-network-based model successfully predicted the sample type with accuracies of 89% and 97%, respectively. Conclusions: Because of the high recognition accuracies and the small amount of computing resources required for these analyses, our platform can be a high precision conventional cell classification system for hPSCs. (C) 2020, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.

关键词： lectin microarray Linear classification Neural network Artificial intelligence Pluripotent stem cells

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Glycome profiling by lectin microarray reveals dynamic glycan alterations during epidermal stem cell aging

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AGING CELL 2020年第8期19卷 e13190-e13190页

作者： Oinam, Lalhaba Changarathil, Gopakumar Raja, Erna Ngo, Yen Xuan Tateno, Hiroaki Sada, Aiko Yanagisawa, Hiromi Univ Tsukuba Life Sci Ctr Survival Dynam Tsukuba Adv Res Alliance TARA Tsukuba Ibaraki Japan Univ Tsukuba Sch Integrat & Global Majors PhD Program Human Biol Tsukuba Ibaraki Japan Univ Tsukuba Grad Sch Comprehens Human Sci Tsukuba Ibaraki Japan Kumamoto Univ Int Res Ctr Med Sci IRCMS Kumamoto Japan Natl Inst Adv Ind Sci & Technol Cellular & Mol Biotechnol Res Inst Tsukuba Cent 61-1-1 Higashi Tsukuba Ibaraki 3058566 Japan Univ Tsukuba Fac Med Tsukuba Ibaraki Japan

Aging in the epidermis is marked by a gradual decline in barrier function, impaired wound healing, hair loss, and an increased risk of cancer. This could be due to age-related changes in the properties of epidermal stem cells and defective interactions with their microenvironment. Currently, no biochemical tools are available to detect and evaluate the aging of epidermal stem cells. The cellular glycosylation is involved in cell-cell communications and cell-matrix adhesions in various physiological and pathological conditions. Here, we explored the changes of glycans in epidermal stem cells as a potential biomarker of aging. Using lectin microarray, we performed a comprehensive glycan profiling of freshly isolated epidermal stem cells from young and old mouse skin. Epidermal stem cells exhibited a significant difference in glycan profiles between young and old mice. In particular, the binding of a mannose-binder rHeltuba was decreased in old epidermal stem cells, whereas that of an alpha 2-3Sia-binder rGal8N increased. These glycan changes were accompanied by upregulation of sialyltransferase,St3gal2andSt6gal1and mannosidaseMan1agenes in old epidermal stem cells. The modification of cell surface glycans by overexpressing these glycogenes leads to a defect in the regenerative ability of epidermal stem cells in culture. Hence, our study suggests the age-related global alterations in cellular glycosylation patterns and its potential contribution to the stem cell function. These glycan modifications detected by lectins may serve as molecular markers for aging, and further functional studies will lead us to a better understanding of the process of skin aging.

关键词： epidermal stem cells glycosylation lectin microarray mannose sialylation skin aging stem cell aging

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Fucosylation is associated with the malignant transformation of intraductal papillary mucinous neoplasms: a lectin microarray-based study

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SURGERY TODAY 2016年第10期46卷 1217-1223页

作者： Watanabe, Kiminori Ohta, Masayuki Yada, Kazuhiro Komori, Yoko Iwashita, Yukio Kashima, Kenji Inomata, Masafumi Oita Univ Fac Med Dept Gastroenterol & Pediat Surg Hasama Machi Oita 8795593 Japan Oita Prefectural Hosp Dept Pathol Oita Japan

Intraductal papillary mucinous neoplasm (IPMN) is an intraductal mucin-producing pancreatic neoplasm with the potential for malignant transformation. Changes in glycans expressed on the cell surface and glycotransferases play important roles in malignant transformation. We conducted this study to analyze glycan alterations in IPMNs by using a lectin microarray and to identify the factors associated with altered glycans and their relationships with malignant transformation. Using a lectin microarray, we evaluated glycan expression in 22 samples of IPMN with carcinoma, obtained from curative resections performed in our department. We also used immunohistochemistry to investigate fucosyltransferase 8 (Fut 8) protein expression, which is associated with glycan alterations in IPMNs. The lectin microarray demonstrated that only two lectins, Aleuria aurantia lectin (AAL) and Aspergillus oryzae l-fucose-specific lectin (AOL), which bind to fucose, exhibited significant sequential increases from normal pancreatic duct to adenoma and carcinoma. Similarly, Fut 8 protein expression, which is associated with AAL and AOL, sequentially and significantly increased from the normal pancreatic duct to adenoma and carcinoma. lectin microarray analysis suggested that fucosylation is associated with the malignant transformation of IPMNs.

关键词： IPMN Fucosylation lectin microarray AAL AOL

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Combinatorial Approach with Mass Spectrometry and lectin microarray Dissected Site-Specific Glycostem and Glycoleaf Features of the Virion-Derived Spike Protein of Ancestral and γ Variant SARS-CoV-2 Strains

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JOURNAL OF PROTEOME RESEARCH 2024年第4期23卷 1408-1419页

作者： Hiono, Takahiro Sakaue, Hiroaki Tomioka, Azusa Kaji, Hiroyuki Sasaki, Michihito Orba, Yasuko Sawa, Hirofumi Kuno, Atsushi Natl Inst Adv Ind Sci & Technol Cellular & Mol Biotechnol Res Inst Mol & Cellular Glycoprote Res Grp Tsukuba Ibaraki 3058565 Japan Hokkaido Univ One Hlth Res Ctr Sapporo Hokkaido 0600818 Japan Hokkaido Univ Fac Vet Med Dept Dis Control Lab Microbiol Sapporo Hokkaido 0600818 Japan Hokkaido Univ Int Inst Zoonosis Control Int Collaborat Unit Sapporo Hokkaido 0010020 Japan Hokkaido Univ Inst Vaccine Res & Dev HU IVReD Sapporo Hokkaido 0010021 Japan Hokkaido Univ Int Inst Zoonosis Control Div Mol Pathobiol Sapporo Hokkaido 0010020 Japan

The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has impacted public health globally. As the glycosylation of viral envelope glycoproteins is strongly associated with their immunogenicity, intensive studies have been conducted on the glycans of the glycoprotein of SARS-CoV-2, the spike (S) protein. Here, we conducted intensive glycoproteomic analyses of the SARS-CoV-2 S protein of ancestral and gamma-variant strains using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarrays (LMA). Our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, identified 1448 (ancestral strain) and 1785 (gamma-variant strain) site-specific glycan compositions, respectively. Asparagine at amino acid position 20 (N20) is mainly glycosylated within two successive potential glycosylation sites, N17 and N20, of the gamma-variant S protein;however, we found low-frequency glycosylation at N17. Our novel approaches, glycostem mapping and glycoleaf scoring, also illustrate the moderately branched/extended, highly fucosylated, and less sialylated natures of the glycoforms of S proteins. Subsequent LMA analysis emphasized the intensive end-capping of glycans by Lewis fucoses, which complemented the glycoproteomic features. These results illustrate the high-resolution glycoproteomic features of the SARS-CoV-2 S protein, contributing to vaccine design and understanding of viral protein synthesis.

关键词： SARS-CoV-2 COVID-19 spike protein glycosylation glycoproteomics lectin microarray mass spectrometry

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