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lectin microarray based glycan profiling of exosomes for dynamic monitoring of colorectal cancer progression

ANALYTICA CHIMICA ACTA 2024年 1316卷 342819页

作者： Sun, Xudong Chen, Bowen Shan, Yongjie Jian, Minghong Wang, Zhenxin Chinese Acad Sci Changchun Inst Appl Chem State Key Lab Electroanalyt Chem Changchun 130022 Peoples R China Univ Sci & Technol China Sch Appl Chem & Engn Hefei 230026 Peoples R China First Hosp Jilin Univ Gen Surg Ctr Dept Thyroid Surg Changchun 130021 Peoples R China Chinese Acad Sci Changchun Inst Appl Chem Natl Analyt Res Ctr Electrochem & Spect Changchun 130022 Peoples R China Chinese Acad Sci Changchun Inst Appl Chem 5625 Ren Min St Changchun 130022 Peoples R China

Background: Exosomes, as emerging biomarkers in liquid biopsies in recent years, offer profound insights into cancer diagnostics due to their unique molecular signatures. The glycosylation profiles of exosomes have emerged as potential biomarkers, offering a novel and less invasive method for cancer diagnosis and monitoring. Colorectal cancer (CRC) represents a substantial global health challenge and burden. Thus there is a great need for the aberrant glycosylation patterns on the surface of CRC cell -derived exosomes, proposing them as potential biomarkers for tumor characterization. Results: The interactions of 27 lectins with exosomes from three CRC cell lines (SW480, SW620, HCT116) and one normal colon epithelial cell line (NCM460) have been analyzed by the lectin microarray. The result indicates that Ulex Europaeus Agglutinin I (UEA-I) exhibits high affinity and specificity towards exosomes derived from SW480 cells. The expression of glycosylation related genes within cells has been analyzed by high -throughput quantitative polymerase chain reaction (HT-qPCR). The experimental result of HT-qPCR is consistent with that of lectin microarray. Moreover, the limit of detection (LOD) of UEA-I microarray is calculated to be as low as 2.7 x 10 5 extracellular vehicles (EVs) mL -1 (three times standard deviation (3 sigma) of blank sample). The UEA-I microarray has been successfully utilized to dynamically monitor the progression of tumors in mice -bearing SW480 CRC subtype, applicable in tumor sizes ranging from 2 mm to 20 mm in diameter. Significance: The results reveal that glycan expression pattern of exosome is linked to specific CRC subtypes, and regulated by glycosyltransferase and glycosidase genes of mother cells. Our findings illuminate the potential of glycosylation molecules on the surface of exosomes as reliable biomarkers for diagnosis of tumor at early stage and monitoring of cancer progression.

关键词： lectin microarray Glycosylation Exosome Colorectal cancer

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Inter-tissue glycan heterogeneity: site-specific glycoform analysis of mouse tissue N-glycoproteomes using MS1-based glycopeptide detection method assisted by lectin microarray

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ANALYTICAL AND BIOANALYTICAL CHEMISTRY 2025年第5期417卷 973-988页

作者： Nagai-Okatani, Chiaki Tomioka, Azusa Tominaga, Daisuke Sakaue, Hiroaki Kuno, Atsushi Kaji, Hiroyuki Natl Inst Adv Ind Sci & Technol Cellular & Mol Biotechnol Res Inst Mol & Cellular Glycoprote Res Grp Tsukuba Ibaraki 3058565 Japan Nagoya Univ Inst Glycocore Res iGCORE Furo Cho Nagoya Aichi 4648601 Japan

To understand the biological and pathological functions of protein glycosylation, the glycan heterogeneities for each glycosite in a single glycoprotein need to be elucidated depending on the type and status of cells. For this aim, a reliable strategy is needed to compare site-specific glycoforms of multiple glycoprotein samples in a comprehensive manner. To analyze this "inter-heterogeneity" of samples, we previously introduced an MS1-based glycopeptide detection method, "Glyco-RIDGE." Here, to elucidate inter-tissue glycan heterogeneities, this estimation method was applied to site-specific N-glycoforms of glycoproteins from six normal mouse tissues (liver, kidney, spleen, pancreas, stomach, and testis). The analyses of desialylated glycopeptides estimated 11,325 site-specific N-glycoforms with 239 glycan compositions at 1260 sites (1122 non-redundant core peptides) in 800 glycoproteins, revealing inter-tissue differences in macro-, micro-, and meta-glycan heterogeneities. To obtain detailed information on their glycan features and tissue distribution, the Glyco-RIDGE results were compared with laser microdissection-assisted lectin microarray (LMD-LMA)-based mouse tissue glycome mapping data deposited on LM-GlycomeAtlas, as well as MS-based mouse tissue glycome data deposited on GlycomeAtlas. This integrated approach supported the certainty of Glyco-RIDGE results and suggested that inter-tissue differences exist in glycan motifs, such as alpha-galactose and bisecting N-acetylglucosamine, in both whole tissue glycomes and specific glycoproteins, Anpep and Lamc1. In addition, the comparison with LMD-LMA-based tissue glycome mapping data suggested the possibility of estimating the tissue distribution of the assigned glycans and glycopeptides. Taken together, these findings demonstrate the utility of an integrated approach using MS assisted by LMA for large-scale analyses.

关键词： Glycoproteomics Site-specific glycoform Glycan heterogeneity Mass spectrometry Tissue glycome mapping lectin microarray

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Glycomic profiling of parathyroid neoplasms via lectin microarray analysis

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ENDOCRINE 2025年第3期87卷 1224-1234页

作者： Zheng, Qingyuan Cui, Ming Xiao, Jinheng Yang, Sen Chen, Tianqi Shi, Yanan Hu, Ya Liao, Quan Chinese Acad Med Sci & Peking Union Med Coll Peking Union Med Coll Hosp Dept Gen Surg State Key Lab Complex Severe & Rare Dis Beijing Peoples R China Chinese Acad Med Sci & Peking Union Med Coll Peking Union Med Coll Hosp Inst Clin Med Biomed EngnFac Natl Infrastruct Translat Med Beijing Peoples R China

PurposeParathyroid carcinoma (PC) is a rare malignancy with a poor prognosis. Diagnosis of PC is often difficult in clinical practice and efficient diagnostic markers are still needed for differential diagnosis. Aberrant glycosylations of glycoproteins were identified with lectin microarray in various cancers, while relevant information is lacking in PC. MethodsIn this study, 8 PC and 6 parathyroid adenoma (PA) tissues were assessed using a microarray consisting of 70 lectins. Overall lectin-specific glycosylation patterns were compared between PA and PC tissues. lectins with significant differential response between PC and PA were further validated by lectin histochemistry. ResultsThe difference in signal intensities was found in 71.4% (50/70) of the lectins between the two groups (P < 0.05). The vast majority of PCs had higher intensity signals than PAs (PCs vs. PAs, ratio >1) and amaranthus caudatus (ACL) showed the most significantly different response between them (ratio = 2.45). lectin histochemistry further confirmed higher ACL intensity in PCs than in PAs. The differentially expressed glycans in PC tissues were primarily glucose, mannose, and galactose-based. ConclusionPC presented unique glycomic features and ACL may serve as a candidate diagnostic marker for PC.

关键词： Parathyroid carcinoma Parathyroid tumor Glycosylation lectin microarray Biomarker

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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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Changes in serum immunoglobulin G glycosylation patterns for antiphospholipid syndrome patients with lectin microarray

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SCANDINAVIAN JOURNAL OF IMMUNOLOGY 2024年第6期99卷 e13366-e13366页

作者： Wang, Yifei Li, Siting Meng, Jingjing Yu, Rui Wang, Qian Tian, Xinping Li, Mengtao Zeng, Xiaofeng Zhao, Jiulang Hu, Chaojun Chinese Acad Med Sci & Peking Union Med Coll Dept Rheumatol & Clin Immunol Beijing Peoples R China Minist Sci & Technol Natl Clin Res Ctr Dermatol & Immunol Dis NCRC DID Beijing Peoples R China Minist Educ Key Lab Rheumatol & Clin Immunol Beijing Peoples R China Zhengzhou Univ Affiliated Hosp 5 Dept Clin Lab Zhengzhou Peoples R China Minist Educ Key Lab Rheumatol & Clin Immunol 1 Shuaifuyuan Beijing 100730 Peoples R China

Antiphospholipid syndrome is a rare autoimmune disease characterized by persistent antiphospholipid antibodies. Immunoglobulin G plays a vital role in disease progression, with its structure and function affected by glycosylation. We aimed to investigate the changes in the serum immunoglobulin G glycosylation pattern in antiphospholipid syndrome patients. We applied lectin microarray on samples from 178 antiphospholipid syndrome patients, 135 disease controls (including Takayasu arteritis, rheumatoid arthritis and cardiovascular disease) and 100 healthy controls. lectin blots were performed for validation of significant differences. Here, we show an increased immunoglobulin G-binding level of soybean agglutinin (p = 0.047, preferring N-acetylgalactosamine) in antiphospholipid syndrome patients compared with healthy and disease controls. Additionally, the immunoglobulin G from antiphospholipid syndrome patients diagnosed with pregnancy events had lower levels of fucosylation (p = 0.001, recognized by Lotus tetragonolobus) and sialylation (p = 0.030, recognized by Sambucus nigra I) than those with simple thrombotic events. These results suggest the unique serum immunoglobulin G glycosylation profile of antiphospholipid syndrome patients, which may inform future studies to design biomarkers for more accurate diagnosis of antiphospholipid syndrome and even for the prediction of clinical symptoms in patients. Serum samples from antiphospholipid syndrome patients were analysed by lectin microarray and validated by lectin blot. We found the unique expression of sugars binding SBA, LTL and SNA-I compared with the control ***

关键词： antiphospholipid syndrome immunoglobulin G lectin microarray

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A tailored lectin microarray for rapid glycan profiling of therapeutic monoclonal antibodies

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MABS 2024年第1期16卷 2304268页

作者： Luo, Shen Zhang, Baolin Ctr Drug Evaluat & Res FDA Off Pharmaceut Qual Silver Spring MD USA FDA Ctr Drug Evaluat & Res Off Pharmaceut Qual Silver Spring MD 20993 USA

Glycosylation plays a crucial role in determining the quality and efficacy of therapeutic antibodies. This necessitates a thorough analysis and monitoring process to ensure consistent product quality during manufacturing. In this study, we introduce a custom-designed lectin microarray featuring nine distinct lectins: rPhoSL, rOTH3, RCA120, rMan2, MAL_I, rPSL1a, PHAE, rMOA, and PHAL. These lectins have been specifically tailored to selectively bind to common N-glycan epitopes found in therapeutic IgG antibodies. By utilizing intact glycoprotein samples, our nine-lectin microarray provides a high-throughput platform for rapid glycan profiling, enabling comparative analysis of glycosylation patterns. Our results demonstrate the practical utility of this microarray in assessing glycosylation across various manufacturing batches or between biosimilar and innovator products. This capacity empowers informed decision-making in the development and production of therapeutic antibodies.

关键词： Bispecific antibody glycan profile glycosylation lectin microarray monoclonal antibody product quality therapeutic antibodies FDA approval

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LM-GlycomeAtlas Ver. 2.0: An Integrated Visualization for lectin microarray-based Mouse Tissue Glycome Mapping Data with lectin Histochemistry

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JOURNAL OF PROTEOME RESEARCH 2021年第4期20卷 2069-2075页

作者： Nagai-Okatani, Chiaki Zou, Xia Fujita, Noriaki Sogabe, Isami Arakawa, Kouiti Nagai, Misugi Angata, Kiyohiko Zhang, Yan Aoki-Kinoshita, Kiyoko F. Kuno, Atsushi Shanghai Jiao Tong Univ Shanghai Ctr Syst Biomed Minist Educ Key Lab Syst Biomed Shanghai 200240 Peoples R China Natl Inst Adv Ind Sci & Technol Cellular & Mol Biotechnol Res Inst Mol & Cellular Glycoprote Res Grp Tsukuba Ibaraki 3058565 Japan Soka Univ Glycan & Life Sci Integrat Ctr GaLSIC Fac Sci & Engn Hachioji Tokyo 1928577 Japan

Laser microdissection-assisted lectin microarray has been used to obtain quantitative and qualitative information on glycans on proteins expressed in microscopic regions of formalin-fixed paraffin-embedded tissue sections. For the effective visualization of this "tissue glycome mapping" data, a novel online tool, LM-GlycomeAtlas (https://***/lm glycomeadas/index), was launched in the freely available glycoscience portal, the GlyCosmos Portal (haps://*** ). In LM-GlycomeAtlas Version 1.0, nine tissues from normal mice were used to provide one data set of glycomic profiles. Here we introduce an updated version of LM-GlycomeAtlas, which includes more spatial information. We designed it to deposit multiple data sets of glycomic profiles with high-resolution histological images, which included staining images with multiple lectins on the array. The additionally implemented interfaces allow users to display multiple histological images of interest (e.g., diseased and normal mice), thereby facilitating the evaluation of tissue glycomic profiling and glyco-pathological analysis. Using these updated interfaces, 451 glycomic profiling data and 42 histological images obtained from 14 tissues of normal and diseased mice were successfully visualized. By easy integration with other tools for glycoproteomic data and protein glycosylation machinery, LM-GlycomeAtlas will be one of the most valuable open resources that contribute to both glycoscience and proteomics communities.

关键词： glycoinformatics web tool endogenous glycoprotein tissue glycome mapping formalin-fixed paraffin-embedded tissue section laser microdissection lectin microarray histochemistiy

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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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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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