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

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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Glycan analysis of therapeutic glycoproteins

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MABS 2016年第2期8卷 205-215页

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

Therapeutic monoclonal antibodies (mAbs) are glycoproteins produced by living cell systems. The glycan moieties attached to the proteins can directly affect protein stability, bioactivity, and immunogenicity. Therefore, glycan variants of a glycoprotein product must be adequately analyzed and controlled to ensure product quality. However, the inherent complexity of protein glycosylation poses a daunting analytical challenge. This review provides an update of recent advances in glycan analysis, including the potential utility of lectin-based microarray for high throughput glycan profiling. Emphasis is placed on comparison of the major types of analytics for use in determining unique glycan features such as glycosylation site, glycan structure, and content.

关键词： lectin microarray Analytics glycosylation biopharmaceuticals monoclonal antibodies therapeutic proteins glycan profiling

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Alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV

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GLYCOCONJUGATE JOURNAL 2016年第2期33卷 125-136页

作者： Qin, Yannan Zhong, Yaogang Ma, Tianran Wu, Fei Wu, Haoxiang Yu, Hanjie Huang, Chen Li, Zheng Xi An Jiao Tong Univ Dept Cell Biol & Genet Key Lab Environm & Genes Related Dis Minist Educ ChinaSch Basic Med SciHlth Sci Ctr Xian 710061 Peoples R China NW Univ Xian Coll Life Sci Lab Funct Glyc Xian 710069 Peoples R China

The incidence of hepatocellular carcinoma (HCC) is closely correlated with hepatitis B virus (HBV)-induced liver cirrhosis. Structural changes in the glycans of serum and tissue proteins are reliable indicators of liver damage. However, little is known about the alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV infection. This study compared the differential expression of liver glycopatterns in 7 sets of normal pericarcinomatous tissues (PCTs), cirrhotic, and tumor tissues from patients with liver cirrhosis and HCC induced by HBV using lectin microarrays. Fluorescence-based lectin histochemistry and lectin blotting were further utilized to validate and assess the expression and distribution of certain glycans in 9 sets of corresponding liver tissue sections. Eight lectins (e.g., Jacalin and AAL) revealed significant difference in cirrhotic tissues versus PCTs. Eleven lectins (e.g., EEL and SJA) showed significant alteration during cirrhotic and tumor progression. The expression of Gala1-3(Fuca1-2) Gal (EEL) and fucosyltransferase 1 was mainly increasing in the cytoplasm of hepatocytes during PCTs-cirrhotic-tumor tissues progression, while the expression of T antigen (ACA and PNA) was decreased sharply in cytoplasm of tumor hepatocytes. Understanding the precision alteration of liver glycopatterns related to the development of hepatitis, cirrhosis, and tumor induced by HBV infection may help elucidate the molecular mechanisms underlying the progression of chronic liver diseases and develop new antineoplastic therapeutic strategies.

关键词： lectin microarray Glycopattern HBV Liver tissue HCC

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Development and Applications of lectins as Biological Tools in Biomedical Research

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MEDICINAL RESEARCH REVIEWS 2016年第2期36卷 221-247页

作者： Dan, Xiuli Liu, Wenlong Ng, Tzi Bun Chinese Univ Hong Kong Fac Med Sch Biomed Sci Shatin Hong Kong Peoples R China Univ Hong Kong LKS Fac Med Dept Orthopaed & Traumatol Pokfulam Hong Kong Peoples R China

As a new and burgeoning area following genomics and proteomics, glycomics has become a hot issue due to its pivotal roles in many physiological and pathological processes. Glycans are much more complicated than genes or proteins since glycans are highly branched and dynamic. Antibodies and lectins are the two major molecular tools applied for glycan profiling. Though the study of antibodies and lectins started at almost the same time in 1880s, lectins gained much less attention than the antibodies until recent decades when the importance and difficulties of glycomics were realized. The present review summarizes the discovery history of lectins and their biological functions with a special emphasis on their various applications as biological tools. Both older techniques that had been developed in the last century and new technologies developed in recent years, especially lectin microarrays and lectin-based biosensors, are included in this account.

关键词： lectin hemagglutinin lectin histochemistry lectin microarray lectin-based biosensor

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N- and O-glycan cell surface protein modifications associated with cellular senescence and human aging

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CELL AND BIOSCIENCE 2016年第1期6卷 14-14页

作者： Itakura, Yoko Sasaki, Norihiko Kami, Daisuke Gojo, Satoshi Umezawa, Akihiro Toyoda, Masashi Tokyo Metropolitan Inst Gerontol Res Team Geriatr Med Vasc Med Itabashi Ku 35-2 Sakae Cho Tokyo 1730015 Japan Kyoto Prefectural Univ Med Dept Regenerat Med Kamigyo Ku 465 Kajii Cho Kyoto 6028566 Japan Natl Res Inst Child Hlth & Dev Dept Reprod Biol Setagaya Ku 2-10-1 Okura Tokyo 1578535 Japan

Background: Glycans play essential roles in biological functions such as differentiation and cancer. Recently, glycans have been considered as biomarkers for physiological aging. However, details regarding the specific glycans involved are limited. Here, we investigated cellular senescence- and human aging-dependent glycan changes in human diploid fibroblasts derived from differently aged skin donors using a lectin microarray. Results: We found that alpha 2-6sialylated glycans in particular differed between elderly- and fetus-derived cells at early passage. However, both cell types exhibited sequentially decreasing alpha 2-3sialylated O-glycan structures during the cellular senescence process and showed similar overall glycan profiles. Conclusions: We observed a senescence-associated decrease in sialylation and increase in galactose exposure. Therefore, glycan profiling using lectin microarrays might be useful for the characterization of biomarkers of aging.

关键词： Cellular senescence Human aging Glycan lectin microarray

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

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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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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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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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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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