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An improved evanescent fluorescence scanner suitable for high-resolution glycome mapping of formalin-fixed paraffin-embedded tissue sections

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ANALYTICAL AND BIOANALYTICAL CHEMISTRY 2023年第28期415卷 6975-6984页

作者： Boottanun, Patcharaporn Nagai-Okatani, Chiaki Nagai, Misugi Ungkulpasvich, Umbhorn Yamane, Shinjiro Yamada, Masao Kuno, Atsushi Natl Inst Adv Ind Sci & Technol Cellular & Mol Biotechnol Res Inst Mol & Cellular Glycoprote Res Grp Tsukuba Ibaraki 3058565 Japan GlycoTechnica Ltd 101 Hiranobiru3 5-28-6 Utsukushigaoka Aoba Ku Yokohama Kanagawa 2250002 Japan EMUKK LLC 2-21-19 Matsunoki Kuwana Mie 5110902 Japan

lectin microarray (LMA) is a high-throughput platform that enables the rapid and sensitive analysis of N- and O-glycans attached to glycoproteins in biological samples, including formalin-fixed paraffin-embedded (FFPE) tissue sections. Here, we evaluated the sensitivity of the advanced scanner based on the evanescent-field fluorescence principle, which is equipped with a 1x infinity correction optical system and a high-end complementary metal-oxide semiconductor (CMOS) image sensor in digital binning mode. Using various glycoprotein samples, we estimated that the mGSR1200-CMOS scanner has at least fourfold higher sensitivity for the lower limit of linearity range than that of a previous charge-coupled device scanner (mGSR1200). A subsequent sensitivity test using HEK293T cell lysates demonstrated that cell glycomic profiling could be performed with only three cells, which has the potential for the glycomic profiling of cell subpopulations. Thus, we examined its application in tissue glycome mapping, as indicated in the online LM-GlycomeAtlas database. To achieve fine glycome mapping, we refined the laser microdissection-assisted LMA procedure to analyze FFPE tissue sections. In this protocol, it was sufficient to collect 0.1 mm(2) of each of the tissue fragments from 5-mu m-thick sections, which differentiated the glycomic profile between the glomerulus and renal tubules of a normal mouse kidney. In conclusion, the improved LMA enables high-resolution spatial analysis, which expands the possibilities of its application classifying cell subpopulations in clinical FFPE tissue specimens. This will be used in the discovery phase for the development of novel glyco-biomarkers and therapeutic targets, and to expand the range of target diseases.

关键词： lectin microarray Evanescent fluorescence Glycan profiling Endogenous glycoproteins Tissue sections Cell subpopulations

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Features, modulation and analysis of glycosylation patterns of therapeutic recombinant immunoglobulin A

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BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS 2022年第2期38卷 247-269页

作者： Argentova, Victoria Aliev, Teimur Dolgikh, Dmitry Pakanova, Zuzana Katrlik, Jaroslav Kirpichnikov, Mikhail Lomonosov Moscow State Univ Sch Biol Dept Bioengn Moscow Russia Lomonosov Moscow State Univ Sch Chem Dept Chem Enzymol Moscow Russia Russian Acad Sci Shemyakin Ovchinnikov Inst Bioorgan Chem Moscow Russia Slovak Acad Sci Inst Chem Bratislava Slovakia

Increasing the production of recombinant antibodies while ensuring high and stable protein quality remains a challenge in mammalian cell culture. This review is devoted to advances in the field of obtaining stable and optimal glycosylation of therapeutic antibodies based on IgA, as well as the subsequent issues of glycosylation control of glycoproteins during their production. Current studies also demonstrate a general need for a more fundamental understanding of the use of CHO cell-based producer cell lines, through which the glycoprofile of therapeutic IgA antibodies is produced and the dependence of glycosylation on culture conditions could be controlled. Optimization of glycosylation improves the therapeutic efficacy and can expand the possibilities for the creation of highly effective glycoprotein therapeutic drugs. Current status and trends in glycan analysis of therapeutic IgA, dominantly based on mass spectrometry and lectin microarrays are herein summarised as well.

关键词： Antibodies IgA glycosylation mass spectrometry lectin microarray

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Temporal alterations in the bovine buttermilk glycome from parturition to milk maturation

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FOOD CHEMISTRY 2016年 211卷 329-338页

作者： Ross, Sarah A. Gerlach, Jared Q. Gill, Satbir K. Lane, Jonathan A. Kilcoyne, Michelle Hickey, Rita M. Joshi, Lokesh Teagasc Food Res Ctr Moorepk Fermoy Cork Ireland Natl Univ Ireland Galway Glycosci Grp Natl Ctr Biomed Engn Sci Galway Ireland Natl Univ Ireland Galway Sch Nat Sci Carbohydrate Signalling Grp Microbiol Galway Ireland

The bovine milk fat globule membrane (MFGM) has many associated biological activities, many of which are linked with specific carbohydrate structures of MFGM glycoconjugates. Bovine buttermilk is a commercially viable source of MFGM and is an under-valued by-product of butter making. However, the changes in buttermilk glycosylation over the course of lactation have not been extensively investigated. In this study, buttermilk was generated from three individual multiparous cows at 13 time points over the first three months of lactation. Buttermilk glycosylation was profiled using lectin microarrays and lectin blotting. Suggested differences in glycosylation, including N-glycosylation, sialylation and fucosylation, were observed between early and late time points and between individual animals. Overall, these data suggest temporal changes in the glycosylation of buttermilk proteins which may have an important impact on commercial isolation of glycosylated ingredients. (C) 2016 Elsevier Ltd. All rights reserved.

关键词： Milk fat globule membrane Glycosylation Glycoconjugates lectin microarray Buttermilk

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Glycoprofiling as a novel tool in serological assays of systemic sclerosis: A comparative study with three bioanalytical methods

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ANALYTICA CHIMICA ACTA 2015年第1期853卷 555-562页

作者： Klukova, Ludmila Bertok, Tomas Petrikova, Miroslava Sediva, Alena Mislovicova, Danica Katrlik, Jaroslav Vikartovska, Alica Filip, Jaroslav Kasak, Peter Andicsova-Eckstein, Anita Mosnacek, Jaroslav Lukac, Jozef Rovensky, Jozef Imrich, Richard Tkac, Jan Slovak Acad Sci Inst Chem Dept Glycobiotechnol Bratislava 84538 Slovakia Qatar Univ Ctr Adv Mat Doha Qatar Slovak Acad Sci Inst Polymer Dept Biomat Res Bratislava 84541 Slovakia Slovak Acad Sci Inst Polymer Dept Synth & Characterizat Polymers Bratislava 84541 Slovakia Natl Inst Rheumat Dis Piestany 92112 Slovakia Slovak Acad Sci Lab Human Endocrinol Inst Expt Endocrinol Bratislava 83306 Slovakia

Systemic sclerosis (SSc) is an autoimmune disease seriously affecting patient's quality of life. The heterogeneity of the disease also means that identification and subsequent validation of biomarkers of the disease is quite challenging. A fully validated single biomarker for diagnosis, prognosis, disease activity and assessment of response to therapy is not yet available. The main aim of this study was to apply an alternative assay protocol to the immunoassay-based analysis of this disease by employment of sialic acid recognizing lectin Sambucus nigra agglutinin (SNA) to glycoprofile serum samples. To our best knowledge this is the first study describing direct lectin-based glycoprofiling of serum SSc samples. Three different analytical methods for glycoprofiling of serum samples relying on application of lectins are compared here from a bioanalytical point of view including traditional ELISA-like lectin-based method (ELLA), novel fluorescent lectin microarrays and ultrasensitive impedimetric lectin biosensors. Results obtained by all three bioanalytical methods consistently showed differences in the level of sialic acid present on glycoproteins, when serum from healthy people was compared to the one from patients having SSc. Thus, analysis of sialic acid content in human serum could be of a diagnostic value for future detection of SSc, but further work is needed to enhance selectivity of assays for example by glycoprofiling of a fraction of human serum enriched in antibodies for individual diagnostics. (C) 2014 Elsevier B.V. All rights reserved.

关键词： lectin Sialic acid Electrochemical impedance spectroscopy lectin microarray Enzyme-linked lectin assay Systemic sclerosis

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Decreased expression of Bauhinia purpurea lectin is a predictor of gastric cancer recurrence

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SURGERY TODAY 2015年第10期45卷 1299-1306页

作者： Futsukaichi, Takuro Etoh, Tsuyoshi Nakajima, Kentaro Daa, Tsutomu Shiroshita, Hidefumi Shiraishi, Norio Kitano, Seigo Inomata, Masafumi Oita Univ Fac Med Dept Gastroenterol & Pediat Surg Yufu Oita 8795593 Japan Oita Univ Fac Med Dept Diagnost Pathol Oita 87011 Japan Oita Univ Fac Med Ctr Community Med Oita 87011 Japan Oita Univ Oita 87011 Japan

Molecular markers as indicators for gastric cancer recurrence are urgently required. The aim of this study was to identify lectins that can be used to predict gastric cancer recurrence after gastrectomy. We created lectin expression profiles by microarray analysis for 60 patients, who underwent surgery for gastric cancer at the Oita University Hospital between January, 2005 and December, 2007. lectin expression and clinicopathological factors in patients who suffered gastric cancer recurrence and those who did not were compared by univariate and multivariate analyses. Thirteen lectins showed a significant increase in binding to cancer tissues, whereas 11 lectins showed a significant decrease in binding to cancer tissues, when compared with binding to normal epithelia. Multivariate analysis revealed that lymph node metastasis and low Bauhinia purpurea lectin (BPL)-binding signals were independent predictive factors for recurrence. All patients with low BPL expression had significantly worse relapse-free survival than those with high BPL expression. Our results using a novel lectin microarray system provide the first solid evidence that BPL expression is a predictor of gastric cancer recurrence.

关键词： Bauhinia purpurea lectin lectin microarray Gastric cancer Recurrence Glycan

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Glycopattern Alteration of Glycoproteins in Gastrointestinal Cancer Cell Lines and Their Cell-Derived Exosomes

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JOURNAL OF PROTEOME RESEARCH 2022年第8期21卷 1876-1893页

作者： Zhang, Jinyuan Qin, Yannan Jiang, Qiuyu Li, Fang Jing, Xintao Cao, Li Cai, Shuang Wu, Fei Li, Qian Lian, Jiangfang Song, Yongfei Huang, Chen Xi An Jiao Tong Univ Inst Genet & Dev Biol Translat Medcine Inst Xian 710301 Peoples R China Xi An Jiao Tong Univ ffi liated Hosp 2 Dept Oncol Xian 710004 Peoples R China First ffi liated Hosp Xian Med Univ Dept Gastroenterol Xian 710000 Peoples R China Lihuili Hosp Facilitated Ningbo Univ Dept Cardiovasc Ningbo 315211 Peoples R China Ningbo Inst Med & Biomed Engn Combined Innovat Ningbo 315000 Peoples R China

ABSTRACT: Gastrointestinal (GI) cancers constitute the largest portion of all human cancers, and the most prevalent GI cancers in China are colorectal cancer (CRC), gastric cancer (GC), and hepatocellular carcinoma (HCC). Exosomes are nanosized vesicles containing proteins, lipids, glycans, and nucleic acid, which play important roles in the tumor microenvironment and progression. Aberrant glycosylation is closely associated with GI cancers;however, little is known about the glycopattern of the exosomes from GI cancer cells. In this study, glycopatterns of HCC, CRC, and GC cell lines and their exosomes were detected using lectin microarrays. For all exosomes, (GlcNAc beta 1-4)n and Gal beta 1-4GlcNAc (DSA) were the most abundant glycans, but alpha GalNAc and alpha Gal (GSL-II and SBA) were the least. Different cancers had various characteristic glycans in either cells or exosomes. Glycans altered in cell-derived exosomes were not always consistent with the host cells in the same cancer. However, Fuc alpha 1-6GlcNAc (core fucose) and Fuc alpha 1-3(Gal beta 1-4)GlcNAc (AAL) were altered consistently in cells and exosomes although they were decreased in HCC and CRC but increased in GC. The study drew the full-scale glycan fingerprint of cells and exosomes related to GI cancer, which may provide useful information for finding specific biomarkers and exploring the underlying mechanism of glycosylation in exosomes.

关键词： gastrointestinal cancer exosome lectin microarray glycopattern

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Development of M2BPGi: a novel fibrosis serum glyco-biomarker for chronic hepatitis/cirrhosis diagnostics

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EXPERT REVIEW OF PROTEOMICS 2015年第6期12卷 683-693页

作者： Narimatsu, Hisashi Natl Inst Adv Ind Sci & Technol Res Ctr Med Glycosci Tsukuba Ibaraki 3058568 Japan

Many proteins in the living body are glycoproteins, which present glycans linked on their surface. Glycan structures reflect the degree of cell differentiation or canceration and are cell specific. These characteristics are advantageous in the development of various disease biomarkers. Glycoprotein-based biomarkers (glyco-biomarkers) are developed by utilizing the specific changes in the glycan structure on a glycoprotein secreted from the diseased cells of interest. Therefore, quantification of the altered glycan structures is the key to developing a new glyco-biomarker. Glycoscience is a relatively new area of molecular science, and recent advancement of glycotechnologies is remarkable. In the author's institute, new glycoscience technologies have been designed to be efficiently utilized for the development of new diagnostic agents. This paper introduces a strategy for glyco-biomarker development, which was successfully applied in the development of Wisteria floribunda agglutinin-positive Mac-2 binding protein M2BPGi, a liver fibrosis marker now commercially available for clinical use.

关键词： cirrhosis glyco-biomarker glycoscience hepatitis IGOT-LC MS lectin microarray liver fibrosis M2BPGi translational research WFA-positive Mac-2 binding protein

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Differential profiling studies of N-linked glycoproteins in glioblastoma cancer stem cells upon treatment with γ-secretase inhibitor

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PROTEOMICS 2011年第20期11卷 4021-4028页

作者： Dai, Lan Liu, Yashu He, Jintang Flack, Callie G. Talsma, Caroline E. Crowley, Jessica G. Muraszko, Karin M. Fan, Xing Lubman, David M. Univ Michigan Med Ctr Dept Surg Ann Arbor MI 48109 USA Univ Michigan Med Ctr Program Bioinformat Ann Arbor MI 48109 USA Univ Michigan Med Ctr Dept Neurosurg Ann Arbor MI 48109 USA

We have recently demonstrated that Notch pathway blockade by gamma-secretase inhibitor (GSI) depletes cancer stem cells (CSCs) in Glioblastoma Multiforme (GBM) through reduced proliferation and induced apoptosis. However, the detailed mechanism by which the manipulation of Notch signal induces alterations on post-translational modifications such as glycosylation has not been investigated. Herein, we present a differential profiling work to detect the change of glycosylation pattern upon drug treatment in GBM CSCs. Rapid screening of differential cell surface glycan structures has been performed by lectin microarray on live cells followed by the detection of N-linked glycoproteins from cell lysates using multi-lectin chromatography and label-free quantitative mass spectrometry analysis. A total of 51 and 52 glycoproteins were identified in the CSC- and GSI-treated groups, respectively, filtered by a combination of decoy database searching and Trans-Proteomic Pipeline (TPP) processing. Although no significant changes were detected from the lectin microarray experiment, 7 differentially expressed glycoproteins with high confidence were captured after the multi-lectin column including key enzymes involved in glycan processing. Functional annotations of the altered glycoproteins suggest a phenotype transformation of CSCs toward a less tumorigenic form upon GSI treatment.

关键词： Cancer stem cells Gliobastoma Glycoproteomics Label-free lectin microarray Multi-lectin chromatography

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A Systems Biology Approach Identifies FUT8 as a Driver of Melanoma Metastasis

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CANCER CELL 2017年第6期31卷 804-+页

作者： Agrawal, Praveen Fontanals-Cirera, Barbara Sokolova, Elena Jacob, Samson Vaiana, Christopher A. Argibay, Diana Davalos, Veronica McDermott, Meagan Nayak, Shruti Darvishian, Farbod Castillo, Mireia Ueberheide, Beatrix Osman, Iman Fenyo, David Mahal, Lara K. Hernando, Eva NYU Sch Med Dept Pathol New York NY 10016 USA NYU Sch Med Perlmutter Canc Ctr Interdisciplinary Melanoma Cooperat Grp New York NY 10016 USA NYU Dept Chem Inst Biomed Chem New York NY 10003 USA NYU Sch Med Dept Biochem & Mol Pharmacol New York NY 10016 USA NYU Sch Med Inst Syst Genet New York NY 10016 USA NYU Sch Med Dept Dermatol New York NY 10016 USA Mt Sinai Hlth Syst Icahn Sch Med Mt Sinai Dept Pathol New York NY 10029 USA

Association of aberrant glycosylation with melanoma progression is based mainly on analyses of cell lines. Here we present a systems-based study of glycomic changes and corresponding enzymes associated with melanoma metastasis in patient samples. Upregulation of core fucosylation (FUT8) and downregulation of alpha-1,2 fucosylation (FUT1, FUT2) were identified as features of metastatic melanoma. Using both in vitro and in vivo studies, we demonstrate FUT8 is a driver of melanoma metastasis which, when silenced, suppresses invasion and tumor dissemination. Glycoprotein targets of FUT8 were enriched in cell migration proteins including the adhesion molecule L1CAM. Core fucosylation impacted L1CAM cleavage and the ability of L1CAM to support melanoma invasion. FUT8 and its targets represent therapeutic targets in melanoma metastasis.

关键词： primary melanoma metastatic melanoma glycosylation lectin microarray lectin array core fucosylation metastasis glycomics FUT8 L1CAM

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BMP-1-induced GBA1 nuclear accumulation provokes CCN2 mRNA expression via importin-β-mediated nucleocytoplasmic pathway

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JOURNAL OF CELL COMMUNICATION AND SIGNALING 2023年第2期17卷 263-274页

作者： Muromachi, Koichiro Nakano, Rei Fujita-Yoshigaki, Junko Sugiya, Hiroshi Tani-Ishii, Nobuyuki Kanagawa Dent Univ Grad Sch Dent Dept Endodont Yokosuka Kanagawa 2388580 Japan RIKEN Yokohama Inst Ctr Integrat Med Sci Lab Mucosal Immun Yokohama Kanagawa 2300045 Japan Nihon Univ Coll Bioresource Sci Dept Vet Med Lab Vet Radiol Fujisawa Kanagawa 2520880 Japan Nihon Univ Dept Physiol Sch Dent Matsudo Matsudo Chiba 2718587 Japan Japan Anim Specialty Med Inst JASMINE Yokohama Kanagawa 2240001 Japan

Bone morphogenetic protein (BMP)-1 is expressed by odontoblasts in the dentin-pulp complex. Although the functional effects of BMP-1 on the maturation of various preforms of proteins and enzymes involved in initiating mineralization have been widely observed, how BMP-1 affects cellular molecules remains unknown. We performed a comprehensive analysis of BMP-1-altered glycome profiles and subsequent assays to identify the target glycoproteins in human dental pulp cells (hDPCs) by a glycomic approach. In the presence of BMP-1, a lectin microarray analysis and lectin-probed blotting showed that alpha 2,6-sialylation was significantly attenuated in insoluble fractions from hDPCs. Six proteins were identified by a mass spectrometry analysis of alpha 2,6-sialylated glycoproteins purified using a lectin column. Among them, glucosylceramidase (GBA1) was found to accumulate in the nuclei of hDPCs in the presence of BMP-1. Moreover, BMP-1-induced cellular communication network factor (CCN) 2 expression, which is well known as the osteogenesis/chondrogenesis marker, was significantly suppressed in the cells transfected with GBA1 siRNA. Furthermore, importazole, a potent inhibitor of importin-beta-mediated nuclear import significantly suppressed BMP-1-induced GBA1 nuclear accumulation and BMP-1-induced CCN2 mRNA expression, respectively. Thus, BMP-1 facilitates the accumulation of GBA1 in the nucleus through the reduction of alpha 2,6-sialic acid, which potentially contributes to the transcriptional regulation of the CCN2 gene via importin-beta-mediated nuclear import pathway in hDPCs. Our results offer new insights into the role of the BMP-1-GBA1-CCN2 axis in the development, tissue remodeling, and pathology of dental/craniofacial diseases. [GRAPHICS] .

关键词： Glycomics lectin microarray LC-MS MS analysis Sialylation Glycosphingolipid

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