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Glycan profiling analysis using evanescent-field fluorescence-assisted lectin array: Importance of sugar recognition for cellular uptake of exosomes from mesenchymal stem cells

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2017年第3期491卷 701-707页

作者： Shimoda, Asako Tahara, Yoshiro Sawada, Shin-ichi Sasaki, Yoshihiro Akiyoshi, Kazunari Kyoto Univ Dept Polymer Chem Grad Sch Engn Nishikyo Ku Kyoto 6158510 Japan Japan Sci & Technol Agcy JST Exploratory Res Adv Technol ERATO Bionanotransporter Project Katsura Inttech CtrNishikyo Ku Kyoto 6158530 Japan

Studies involving the functional analysis of exosomal contents including proteins, DNA, and RNA have been reported. Most membrane proteins and lipids are glycosylated, which controls their physical properties and functions, but little is known about glycans on exosomes owing to the difficulty of analysing them. To shed light on these issues, we collected exosomes from mesenchymal stem cells (MSCs) derived from human adipose tissue for glycan profiling using evanescent-field fluorescence assisted lectin array as well as analysis of their uptake in vivo. Initial analyses showed that the mean diameter of the collected exosomes was 178 nm and they presented with typical exosomal and MSC markers. Regarding the glycan profiling, exosomes interacted more strongly than the membrane of the original MSCs did with a range of lectins, especially sialic acid-binding lectins. The findings also showed that cellular exosome uptake involved recognition by HeLa cell-surface-bound sialic acid-binding immunoglobulin (1g)-like lectins (siglecs). Confirming this siglec-related uptake, in vivo experiments involving subcutaneous injection of the fluorescently labelled exosomes into mice showed their transport into lymph nodes and internalization by antigen-presenting cells, particularly those expressing CD11b. Closer analysis revealed the colocalization of the exosomes with siglecs, indicating their involvement in the uptake. These findings provide us with an improved understanding of the importance of exosomal transport and targeting in relation to glycans on exosomal surfaces, potentially enabling us to standardize exosomes when using them for therapeutic purposes.(C) 2017 Elsevier Inc. All rights reserved.

关键词： Exosome Mesenchymal stem cell Glycan lectin array Sialic acid

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lectin array to detect ionizing radiation-induced changes in cell surface glycans

Lectin array to detect ionizing radiation-induced changes in...

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第十届全国化学生物学学术会议

作者： ZHAO Zhenjun MOUTRIE Vaughan SANTOS Daniel MCROBB Lucinda SHI Bingyan STOODLEY Marcus.A Faculty of Medicine and Health Sciences Macquarie University Genesis Cancer Care Macquarie University Hospital

Background: Our goal is to identify specific markers on the endothelial surface of human brain arteriovenous malformations（AVMs） induced by ionizing radiation that could be used as targets to site-specifically deliver thrombotic agents to block blood flow and eliminate the risk of rupture of these abnormal vessels [1]. Ionizing radiation damages cells, alters biochemical pathways and causes various changes in cellular and molecular levels. Some of the molecular changes on endothelial surface may provide novel vascular targets. In this study,we aimed to examine radiation-induced changes in the endothelial surface glycome using a lectin ***: Cultured human cerebral microvascular endothelial cells（hCMEC/D3） were irradiated at different doses（5 Gy, 15 Gy or 25 Gy） using a linear accelerator, or sham-irradiated（controls）. At day 1, 3 and 7 post-radiation,viable cells were gently harvested by 0.05% trypsin and cell suspensions incubated in the wells of the lectin microarray slides（RayBiotech, GA, USA）,which had been coated with 40 different lectins, each at a precise location. The binding of the cells with the lectins was detected under a microscope. Cell binding to a lectin means the corresponding glycan structure is expressed on the surface of the cells. The number of the cells binding to the spot area indicates the level of ***: Overall, cell binding to UEA-1, PHA-E, PHA-L, PNA, AAL, LEA,STL, MAA and DSA was detected. Radiation at 5 Gy and 15 Gy induced binding to DSA at day 1 post-irradiation, and to LEA and AAL at day 3. Binding to PHA-E and PHA-L was up-regulated at day 3 on cells that received 15 and 25 Gy ***:1. The lectin array can detect cell surface glycan expression on intact cells.2. Ionizing radiation can induce the expression of glycans that bind to DSA, LEA and AAL on the surface of brain endothelial cells.3. The radiation-induced glycan may be used as targets for radiation-guided drug delivery.

关键词： lectin array Cell surface glycan Ionizing radiation Site-specific drug delivery Endothelial cells

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The therapeutic capacity of bone marrow MSC-derived extracellular vesicles in Achilles tendon healing is passage-dependent and indicated by specific glycans

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FEBS LETTERS 2022年第8期596卷 1047-1058页

作者： Hayashi, Yuta Yimiti, Dilimulati Sanada, Yohei Ding, Chenyang Omoto, Takenori Ogura, Toshihiko Nakasa, Tomoyuki Ishikawa, Masakazu Hiemori, Keiko Tateno, Hiroaki Miyaki, Shigeru Adachi, Nobuo Hiroshima Univ Grad Sch Biomed & Hlth Sci Dept Orthopaed Surg Hiroshima Japan Hiroshima Univ Grad Sch Biomed & Hlth Sci Dept Musculoskeletal Traumatol & Reconstruct Surg Hiroshima Japan Hiroshima Univ Hosp Med Ctr Translat & Clin Res Hiroshima Japan Natl Inst Adv Ind Sci & Technol Hlth & Med Res Inst Tsukuba Ibaraki Japan Hiroshima Univ Grad Sch Biomed & Hlth Sci Dept Artificial Joints & Biomat Hiroshima Japan Natl Inst Adv Ind Sci & Technol Cellular & Mol Biotechnol Res Inst Tsukuba Ibaraki Japan

The therapeutic potential of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) for various diseases and tissue repair is attracting attention. Here, EVs from conditioned medium of human bone marrow MSCs at passage 5 (P5) and passage 12 (P12) were analysed using mouse Achilles tendon rupture model and lectin microarray. P5 MSC-EVs accelerated Achilles tendon healing compared with P12 MSC-EVs. Fucose-specific lectin TJA-II was indicated as a glycan marker for therapeutic MSC-EVs. The present study demonstrated that early passaged MSC-EVs promote Achilles tendon healing compared with senescent MSC-EVs. Glycans on MSC-EVs might provide useful tools to establish a quality control and isolation system for therapeutic MSC-EVs in regenerative medicine.

关键词： achilles tendon extracellular vesicles glycome lectin array mesenchymal stem cell regenerative medicine tendon healing

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Sweet systems: technologies for glycomic analysis and their integration into systems biology

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CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY 2021年第3期56卷 301-320页

作者： Chen, Shuhui Qin, Rui Mahal, Lara K. NYU Dept Chem New York NY USA Univ Alberta Dept Chem Edmonton AB Canada

Found in virtually every organism, glycans are essential molecules that play important roles in almost every aspect of biology. The composition of glycome, the repertoire of glycans in an organism or a biological sample, is often found altered in many diseases, including cancer, infectious diseases, metabolic and developmental disorders. Understanding how glycosylation and glycomic changes enriches our knowledge of the mechanisms of disease progression and sheds light on the development of novel therapeutics. However, the inherent diversity of glycan structures imposes challenges on the experimental characterization of glycomes. Advances in high-throughput glycomic technologies enable glycomic analysis in a rapid and comprehensive manner. In this review, we discuss the analytical methods currently used in high-throughput glycomics, including mass spectrometry, liquid chromatography and lectin microarray. Concomitant with the technical advances is the integration of glycomics into systems biology in the recent years. Herein we elaborate on some representative works from this recent trend to underline the important role of glycomics in such integrated approaches to disease.

关键词： Glycosylation lectin array lectin microarray systems biology glycome glycomics

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A lectin array analysis for wild-type and α-Gal-knockout pig islets versus healthy human islets

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SURGERY TODAY 2013年第12期43卷 1439-1447页

作者： Miyagawa, Shuji Maeda, Akira Takeishi, Shunsaku Ueno, Takehisa Usui, Noriaki Matsumoto, Shinichi Okitsu, Teru Goto, Masafumi Nagashima, Hiroshi Osaka Univ Grad Sch Med Dept Surg Div Organ Transplantat Suita Osaka 5650871 Japan Moritex Corp Glyc Res Lab Yokohama Kanagawa Japan Natl Ctr Global Hlth & Med Tokyo Japan Univ Tokyo Inst Ind Sci Meguro Ku Tokyo 1538505 Japan Tohoku Univ Int Adv Res & Educ Org Sendai Miyagi 980 Japan Meiji Univ Dept Life Sci Lab Dev Engn Kanagawa Japan

We performed lectin microarray analyses of islets from wild-type (WT) pigs and alpha 1-3galactosyltransferase gene knockout (GKO) pigs and compared the results with the corresponding values for islets from healthy humans. Islets were isolated from the pancreas. After sonication and centrifugation, the proteins in the supernatant from each islet were labeled with Cy3 and applied to a lectin array. Despite negligible expression of the Gal antigen on the adult pig islets (APIs), GKO-islets showed weaker signals, not only for GS-I-B4 but also for PNA, WFA, PTL-I, and GS-I-A4, than the WT islets, indicating reduced contents of alpha-linked GalNAc and Gal beta 1-3GalNAc. In comparing the islets of pigs vs. humans, human islets showed stronger signals for UEA-I, AAL, TJA-II, EEL, WFA, HPA, DBA, SBA and PTL-I, indicating that besides ABO blood type antigens, high levels of fucose and alpha-linked GalNAc are present. On the other hand, the high mannose form was very rich in the APIs. GKO reduced alpha-linked GalNAc, despite negligible expression of the Gal antigen on WT-API. On the other hand, the high-mannose form was richer in both APIs than in healthy human islets. These results provide useful information for future studies.

关键词： Xenotransplantation Glycoantigen lectin array Islets

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Cell surface lectin array: parameters affecting cell glycan signature

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GLYCOCONJUGATE JOURNAL 2013年第3期30卷 195-203页

作者： Landemarre, Ludovic Cancellieri, Perrine Duverger, Eric Univ Orleans GLYcoDIAG F-45067 Orleans 2 France Univ Orleans GlycoBiochim Dept Biol F-45067 Orleans 2 France

Among the "omics", glycomics is one of the most complex fields and needs complementary strategies of analysis to decipher the "glycan dictionary". As an alternative method, which has developed since the beginning of the 21st century, lectin array technology could generate relevant information related to glycan motifs, accessibility and a number of other valuable insights from molecules (purified and non-purified) or cells. Based on a cell line model, this study deals with the key parameters that influence the whole cell surface glycan interaction with lectin arrays and the consequences on the interpretation and reliability of the results. The comparison between the adherent and suspension forms of Chinese Hamster Ovary (CHO) cells, showed respective glycan signatures, which could be inhibited specifically by neoglycoproteins. The modifications of the respective glycan signatures were also revealed according to the detachment modes and cell growth conditions. Finally the power of lectin array technology was highlighted by the possibility of selecting and characterizing a specific clone from the mother cell line, based on the slight difference determination in the respective glycan signatures.

关键词： Carbohydrate Cell lines Glycomics lectin array

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Age-Dependent Glycomic Response to the 2009 Pandemic H1N1 Influenza Virus and Its Association with Disease Severity

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JOURNAL OF PROTEOME RESEARCH 2020年第11期19卷 4486-4495页

作者： Chen, Shuhui Kasper, Brian Zhang, Bin Lashua, Lauren P. Ross, Ted M. Ghedin, Elodie Mahal, Lara K. NYU Dept Chem Biomed Res Inst 4 Washington Pl New York NY 10003 USA Icahn Sch Med Mt Sinai Icahn Inst Genom & Multiscale Biol Dept Genet & Genom Sci Mt Sinai Ctr Transformat Dis Modeling New York NY 10029 USA NYU Dept Biol Ctr Genom & Syst Biol New York NY 10003 USA Univ Georgia Ctr Vaccines & Immunol Athens GA 30602 USA NIAID Syst Genom Sect Lab Parasit Dis NIH Bethesda MD 20894 USA Univ Alberta Dept Chem Edmonton AB T6G 2G2 Canada

Influenza A viruses cause a spectrum of responses, from mild coldlike symptoms to severe respiratory illness and death. Intrinsic host factors, such as age, can influence disease severity. Glycosylation plays a critical role in influenza pathogenesis;however, the molecular drivers of influenza outcomes remain unknown. In this work, we characterized the host glycomic response Refen rice to the H1N1 2009 pandemic influenza A virus (H1N1pdm09) as a function of age-dependent severity in a ferret model. Using our dual-color lectin microarray technology, we examined baseline glycosylation and glycomic response to infection in newly weaned and aged animals, models for young children and the elderly, respectively. Compared to adult uninfected ferrets, we observed higher levels of alpha-2,6-sialosides, the receptor for H1N1pdm09, in newly weaned and aged animals. We also observed age-dependent loss of O-linked alpha-2,3-sialosides. The loss of these highly charged groups may impact viral clearance by mucins, which corresponds to the lower clearance rates observed in aged animals. Upon infection, we observed dramatic changes in the glycomes of aged animals, a population severely impacted by the virus. In contrast, no significant alterations were observed in the newly weaned animals, which show mild to moderate responses to the H1N1pdm09. High mannose, a glycan recently identified as a marker of severity in adult animals, increased with severity in the aged population. However, the response was delayed, in line with the delayed development of pneumonia observed. Overall, our results may help explain the differential susceptibility to influenza A infection and severity observed as a function of age.

关键词： lectin array lectin microarray glycomics influenza high mannose H1N1

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Glycomic analysis of host response reveals high mannose as a key mediator of influenza severity

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2020年第43期117卷 26926-26935页

作者： Heindel, Daniel W. Koppolu, Sujeethraj Zhang, Yue Kasper, Brian Meche, Lawrence Vaiana, Christopher A. Bissel, Stephanie J. Carter, Chalise E. Kelvin, Alyson A. Elaish, Mohamed Lopez-Orozco, Joaquin Zhang, Bin Zhou, Bin Chou, Tsui-Wen Lashua, Lauren Hobman, Tom C. Ross, Ted M. Ghedin, Elodie Mahal, Lara K. NYU Dept Chem Biomed Res Inst New York NY 10003 USA Univ Pittsburgh Sch Med Dept Pathol Pittsburgh PA 15261 USA Univ Georgia Ctr Vaccines & Immunol Athens GA 30602 USA Dalhousie Univ Dept Pediat Halifax NS B3H 4R2 Canada Univ Alberta Dept Cell Biol Edmonton AB T6G 2R7 Canada Cairo Univ Poultry Dis Dept Fac Vet Med Cairo 12211 Egypt Univ Alberta Fac Med & Dent High Content Anal Core Edmonton AB T6G 2R7 Canada Icahn Sch Med Mt Sinai Dept Genet & Genom Sci Mt Sinai Ctr Transformat Dis Modeling Icahn Inst Genom & Multiscale Biol New York NY 10029 USA NYU Dept Biol Ctr Genom & Syst Biol New York NY 10003 USA NYU Sch Global Publ Hlth Dept Epidemiol New York NY 10003 USA NIAID Lab Parasit Dis NIH 9000 Rockville Pike Bethesda MD 20892 USA Univ Alberta Dept Chem Edmonton AB T6G 2G2 Canada

Influenza virus infections cause a wide variety of outcomes, from mild disease to 3 to 5 million cases of severe illness and similar to 290,000 to 645,000 deaths annually worldwide. The molecular mechanisms underlying these disparate outcomes are currently unknown. Glycosylation within the human host plays a critical role in influenza virus biology. However, the impact these modifications have on the severity of influenza disease has not been examined. Herein, we profile the glycomic host responses to influenza virus infection as a function of disease severity using a ferret model and our lectin microarray technology. We identify the glycan epitope high mannose as a marker of influenza virus-induced pathogenesis and severity of disease outcome. Induction of high mannose is dependent upon the unfolded protein response (UPR) pathway, a pathway previously shown to associate with lung damage and severity of influenza virus infection. Also, the mannan-binding lectin (MBL2), an innate immune lectin that negatively impacts influenza outcomes, recognizes influenza virus-infected cells in a high mannose-dependent manner. Together, our data argue that the high mannose motif is an infection-associated molecular pattern on host cells that may guide immune responses leading to the concomitant damage associated with severity.

关键词： lectin microarray MBL2 lectin array mannose binding lectin mannan binding lectin

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Analysis of a lectin microarray identifies altered sialylation of mouse serum glycoproteins induced by whole-body radiation exposure

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JOURNAL OF RADIATION RESEARCH 2019年第2期60卷 189-196页

作者： Iizuka, Daisuke Izumi, Shunsuke Suzuki, Fumio Kamiya, Kenji Natl Inst Quantum & Radiol Sci & Technol Natl Inst Radiol Sci Dept Radiat Effects Res Inage Ku 4-9-1 Anagawa Chiba 2638555 Japan Hiroshima Univ Dept Expt Oncol Res Inst Radiat Biol & Med Minami Ku 1-2-3 Kasumi Hiroshima 7348553 Japan Hiroshima Univ Grad Sch Sci Dept Math & Life Sci 1-3-2 Kagamiyama Higashihiroshima 7398511 Japan Hiroshima Univ Dept Mol Radiobiol Res Inst Radiat Biol & Med Minami Ku 1-2-3 Kasumi Hiroshima 7348553 Japan

Microarrays containing 45 different lectins were analyzed to identify global changes in the glycosylation of serum glycoproteins from mice exposed to whole-body gamma-radiation. The results showed that radiation exposure increased and decreased the relative amounts of alpha-2,3- and alpha-2,6-sialic acids, respectively. The expression of alpha-2,3- and alpha-2,6-sialyltransferase genes in the liver was analyzed to determine whether changes in their expression were responsible for the sialic acid changes. The increase in alpha-2,3-sialic acid correlated with St3gal5 upregulation after radiation exposure;however, a decrease in St6gal1 expression was not observed. Analysis of a PCR array of genes expressed in irradiated mouse livers revealed that irradiation did not alter the expression of most of the included genes. These results suggest that glycomic screening of serum glycoproteins using lectin microarrays can be a powerful tool for identifying radiation-induced changes in the post-translational addition of sugar moieties to proteins. In addition, the results indicate that altered sialylation of glycoproteins may be an initial response to acute radiation exposure.

关键词： lectin array glycosylation adipsin sialic acid sialyltransferase

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Interrogation of Milk-Driven Changes to the Proteome of Intestinal Epithelial Cells by Integrated Proteomics and Glycomics

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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2019年第7期67卷 1902-1917页

作者： Morrin, Sinead T. Owens, Rebecca A. Le Berre, Marie Gerlach, Jared Q. Joshi, Lokesh Bode, Lars Irwin, Jane A. Hickey, Rita M. Teagasc Food Res Ctr Moorepk Fermoy P61C996 Cork Ireland Maynooth Univ Dept Biol Maynooth W23 F2H6 Kildare Ireland Natl Univ Ireland Galway Glycosci Grp Natl Ctr Biomed Engn Sci Adv Glycosci Res Cluster Galway H91TK33 Ireland Univ Calif San Diego Dept Pediat La Jolla CA 92093 USA Univ Calif San Diego Larsson Rosenquist Fdn Mother Milk Infant Ctr Res Excellence La Jolla CA 92093 USA Univ Coll Dublin Sch Vet Med Vet Sci Ctr Dublin D04 V1W8 4 Ireland

Bovine colostrum is a rich source of bioactive components which are important in the development of the intestine, in stimulating gut structure and function and in preparing the gut surface for subsequent colonization of microbes. What is not clear, however, is how colostrum may affect the repertoire of receptors and membrane proteins of the intestinal surface and the post-translational modifications associated with them. In the present work, we aimed to characterize the surface receptor and glycan profile of human HT-29 intestinal cells after exposure to a bovine colostrum fraction (BCF) by means of proteomic and glycomic analyses. Integration of label-free quantitative proteomic analysis and lectin array profiles confirmed that BCF exposure results in changes in the levels of glycoproteins present at the cell surface and also changes to their glycosylation pattern. This study contributes to our understanding of how milk components may regulate intestinal cells and prime them for bacterial interaction.

关键词： bovine colostrum proteome receptor glycomics lectin array milk

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