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Spatiotemporal changes of tissue glycans depending on localization in cardiac aging

REGENERATIVE THERAPY 2023年 22卷 68-78页

作者： Itakura, Yoko Hasegawa, Yasuko Kikkawa, Yurika Murakami, Yuina Sugiura, Kosuke Nagai-Okatani, Chiaki Sasaki, Norihiko Umemura, Mariko Takahashi, Yuji Kimura, Tohru Kuno, Atsushi Ishiwata, Toshiyuki Toyoda, Masashi Tokyo Metropolitan Inst Gerontol Res Team Geriatr Med Vasc Med 35-2 Sakae choItabashi ku Tokyo 1730015 Japan Tokyo Metropolitan Inst Gerontol Div Aging & Carcinogenesis Res Team Geriatr Pathol 35-2 Sakae choItabashi ku Tokyo 1730015 Japan Kitasato Univ Sch Sci Dept Biosci Lab Stem Cell Biol 1-15-1 Kitasato Minami ku Sagamihara Kanagawa 2520373 Japan Tokyo Univ Pharm & Life Sci Sch Life Sci Lab Environm Mol Physiol 1432-1 Horinouchi Hachioji Tokyo 1920392 Japan Natl Inst Adv Ind Sci & Technol Cellular & Mol Biotechnol Res Inst 5 Cent Tsukuba 1-1-1 Higashi Tsukuba Ibaraki 3058565 Japan

Introduction: Heart failure is caused by various factors, making the underlying pathogenic mechanisms difficult to identify. Since cardiovascular disease tends to worsen over time, early diagnosis is key for treatment. In addition, understanding the qualitative changes in the heart associated with aging, where information on the direct influences of aging on cardiovascular disease is limited, would also be useful for treatment and diagnosis. To fill these research gaps, the focus of our study was to detect the structural and functional molecular changes associated with the heart over time, with a focus on glycans, which reflect the type and state of ***: We investigated glycan localization in the cardiac tissue of normal mice and their alterations during aging, using evanescent-field fluorescence-assisted lectin microarray, a technique based on lectin-glycan interaction, and lectin ***: The glycan profiles in the left ventricle showed differences between the luminal side (medial) and wall side (lateral) regions. The medial region was characterized by the presence of sialic acid residues. Moreover, agerelated changes in glycan profiles were observed at a younger age in the medial region. The difference in the age-related decrease in the level of a-galactose stained with Griffonia simplicifolia lectin-IB4 in different regions of the left ventricle suggests spatiotemporal changes in the number of microvessels. Conclusions: The glycan profile, which retains diverse glycan structures, is supported by many cell populations, and maintains cardiac function. With further research, glycan localization and changes have the potential to be developed as a marker of the signs of heart failure. (c) 2022, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://***/licenses/by-nc-nd/ 4.0/).

关键词： Cardiac tissue lectin microarray Aging Glycan profile Microvessels

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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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Alterations of Glycan Composition in Aerobic Granular Sludge during the Adaptation to Seawater Conditions

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ACS ES&T WATER 2023年第1期4卷 279-286页

作者： Chen, Le Min Keisham, Sunanda Tateno, Hiroaki van Ede, Jitske Pronk, Mario van Loosdrecht, Mark C. M. Lin, Yuemei Delft Univ Technol Dept Biotechnol NL-2629 HZ Delft Netherlands Natl Inst Adv Ind Sci & Technol Cellular & Mol Biotechnol Res Inst Tsukuba Ibaraki 3058566 Japan Royal HaskoningDHV NL-3800 AL Amersfoort Netherlands

Bacteria can synthesize a diverse array of glycans, being found attached to proteins and lipids or as loosely associated polysaccharides to the cells. The major challenge in glycan analysis in environmental samples lies in developing high-throughput and comprehensive characterization methodologies to elucidate the structure and monitor the change of the glycan profile, especially in protein glycosylation. To this end, in the current research, the dynamic change of the glycan profile of a few extracellular polymeric substance (EPS) samples was investigated by high-throughput lectin microarray and mass spectrometry, as well as sialylation and sulfation analysis. Those EPS were extracted from aerobic granular sludge collected at different stages during its adaptation to the seawater condition. It was found that there were glycoproteins in all of the EPS samples. In response to the exposure to seawater, the amount of glycoproteins and their glycan diversity displayed an increase during adaptation, followed by a decrease once the granules reached a stable state of adaptation. Information generated sheds light on the approaches to identify and monitor the diversity and dynamic alteration of the glycan profile of the EPS in response to environmental stimuli.

关键词： glycans glycoproteins aerobic granular sludge extracellular polymeric substances lectin microarray

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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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α2,6-Sialylation Is Upregulated in Severe COVID-19, Implicating the Complement Cascade

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ACS INFECTIOUS DISEASES 2022年第11期8卷 2348 - 2361页

作者： Qin, Rui Kurz, Emma Chen, Shuhui Zeck, Briana Chiribogas, Luis Jackson, Dana Herchen, Alex Attia, Tyson Carlock, Michael Rapkiewicz, Amy Bar-Sagi, Dafna Ritchie, Bruce Ross, Ted M. Mahal, Lara K. Univ Alberta Dept Chem Edmonton AB T6G 2G2 Canada NYU Grossman Sch Med Dept Cell Biol New York NY 10016 USA NYU Biomed Res Inst Dept Chem New York NY 10003 USA NYU Langone Ctr Biospecimen Res & Dev New York NY 10016 USA Univ Alberta Hosp Edmonton AB T6G 2B7 Canada Univ Georgia Ctr Vaccines & Immunol Athens GA 30605 USA NYU Long Isl Sch Med Dept Pathol Mineola NY 11501 USA NYU Grossman Sch Med Dept Biochem & Mol Pharmacol New York NY 10016 USA

Better understanding of the molecular mechanisms underlying COVID-19 severity is desperately needed in current times. Although hyper-inflammation drives severe COVID-19, precise mechanisms triggering this cascade and what role glycosylation might play therein are unknown. Here we report the first high-throughput glycomic analysis of COVID-19 plasma samples and autopsy tissues. We find that alpha 2,6-sialylation is upregulated in the plasma of patients with severe COVID-19 and in autopsied lung tissue. This glycan motif is enriched on members of the complement cascade (e.g., C5, C9), which show higher levels of sialylation in severe COVID-19. In the lung tissue, we observe increased complement deposition, associated with elevated alpha 2,6-sialylation levels, corresponding to elevated markers of poor prognosis (IL-6) and fibrotic response. We also observe upregulation of the alpha 2,6-sialylation enzyme ST6GAL1 in patients who succumbed to COVID-19. Our work identifies a heretofore undescribed relationship between sialylation and complement in severe COVID-19, potentially informing future therapeutic development.

关键词： glycosylation sialic acid lectin microarray SARS-CoV-2 complement lectin array

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Metabolism and global protein glycosylation are differentially expressed in healthy and osteoarthritic equine carpal synovial fluid

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EQUINE VETERINARY JOURNAL 2022年第2期54卷 323-333页

作者： Noordwijk, Kira J. Qin, Rui Diaz-Rubio, Maria E. Zhang, Sheng Su, Jin Mahal, Lara K. Reesink, Heidi L. Cornell Univ Coll Vet Med Dept Clin Sci Ithaca NY 14853 USA NYU Dept Chem New York NY USA Univ Alberta Dept Chem Edmonton AB Canada Cornell Univ Inst Biotechnol Prote & Metabol Facil Ithaca NY USA

Background Carpal osteochondral fragmentation and subsequent post-traumatic osteoarthritis (PTOA) are leading causes of wastage in the equine athlete. Identification of synovial fluid biomarkers could contribute to the diagnosis and understanding of osteoarthritis (OA) pathophysiology. Objective The aim of this study was to identify differentially expressed metabolic and glycosylation pathways in synovial fluid from healthy horses and horses with naturally occurring carpal OA. Study design Cross-sectional, in vivo metabolomics and glycomics study. Methods In cohort 1, carpal synovial fluid (n = 12 horses;n = 6 healthy, n = 6 OA) was analysed using high-resolution liquid chromatography mass spectrometry (LC-MS). In cohort 2 (n = 40 horses;n = 20 healthy, n = 20 OA), carpal synovial fluid was analysed using lectin microarrays and a lubricin sandwich ELISA. Results Metabolomic analysis identified >4900 LC-MS features of which 84 identifiable metabolites were differentially expressed (P < .05) between healthy and OA joints, including key pathways related to inflammation (histidine and tryptophan metabolism), oxidative stress (arginine biosynthesis) and collagen metabolism (lysine metabolism). Principle Component Analysis and Partial Least Squares Discriminant Analysis demonstrated separation between healthy and OA synovial fluid. lectin microarrays identified distinct glycosylation patterns between healthy and OA synovial fluid, including increased Core 1/Core 3 O-glycosylation, increased alpha-2,3 sialylation and decreased alpha-1,2 fucosylation in OA. O-glycans predominated over N-glycans in all synovial fluid samples, and synovial fluid lubricin was increased in OA joints as compared to controls. Main limitations The sample size in cohort 1 was limited, and there is inherent variation in severity and duration of joint injury in naturally occurring OA. However, LC-MS identified up to 5000 unique features. Conclusions These data suggest new potential diagnostic and the

关键词： horse glycomics lectin microarray lubricin proteoglycan 4 mass spectrometry metabolomics O‐ glycosylation

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Prevaccination Glycan Markers of Response to an Influenza Vaccine Implicate the Complement Pathway

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JOURNAL OF PROTEOME RESEARCH 2022年第8期21卷 1974-1985页

作者： Qin, Rui Meng, Guanmin Pushalkar, Smruti Carlock, Michael A. Ross, Ted M. Vogel, Christine Mahal, Lara K. Univ Alberta Dept Chem Edmonton AB T6G 2G2 Canada NYU Ctr Genom & Syst Biol Dept Biol New York NY 10003 USA Univ Georgia Ctr Vaccines & Immunol Athens GA 30602 USA

A key to improving vaccine design and vaccination strategy is to understand the mechanism behind the variation of vaccine response with host factors. Glycosylation, a critical modulator of immunity, has no clear role in determining vaccine responses. To gain insight into the association between glycosylation and vaccine-induced antibody levels, we profiled the pre-and postvaccination serum protein glycomes of 160 Caucasian adults receiving the FLUZONE influenza vaccine during the 2019-2020 influenza season using lectin microarray technology. We found that prevaccination levels of Lewis A antigen (Lea) are significantly higher in nonresponders than responders. Glycopro-teomic analysis showed that Lea-bearing proteins are enriched in complement activation pathways, suggesting a potential role of glycosylation in tuning the activities of complement proteins, which may be implicated in mounting vaccine responses. In addition, we observed a postvaccination increase in sialyl Lewis X antigen (sLex) and a decrease in high mannose glycans among high responders, which were not observed in nonresponders. These data suggest that the immune system may actively modulate glycosylation as part of its effort to establish effective protection postvaccination.

关键词： vaccine influenza immunity antibody response glycosylation glycome lectin microarray

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Glycomic Analysis Reveals a Conserved Response to Bacterial Sepsis Induced by Different Bacterial Pathogens

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ACS INFECTIOUS DISEASES 2022年第5期8卷 1075-1085页

作者： Heindel, Daniel W. Chen, Shuhui Aziz, Peter, V Chung, Jonathan Y. Marth, Jamey D. Mahal, Lara K. NYU Biomed Res Inst Dept Chem New York NY 10003 USA SBP Med Discovery Inst La Jolla CA 92037 USA Univ Alberta Dept Chem Edmonton AB T6G 2G2 Canada

Sepsis is an extreme inflammatory response to infection that occurs in the bloodstream and causes damage throughout the body. Glycosylation is known to play a role in immunity and inflammation, but the role of glycans in sepsis is not well-defined. Herein, we profiled the serum glycomes of experimental mouse sepsis models to identify changes induced by 4 different clinical bacterial pathogens (Gram-positive: Streptococcus pneumoniae and Staphylococcus aureus, Gramnegative: Escherichia coli and Salmonella Typhimurium) using our lectin microarray technology. We observed global shifts in the blood sera glycome that were conserved across all four species, regardless of whether they were Gram positive or negative. Bisecting GlcNAc was decreased upon sepsis and a strong increase in core 1/3 O-glycans was observed. lectin blot analysis revealed a high molecular weight protein induced in sepsis by all four bacteria as the major cause of the core 1/3 O-glycan shift. Analysis of this band by mass spectrometry identified interalpha-trypsin inhibitor heavy chains (ITIHs) and fibronectin, both of which are associated with human sepsis. Shifts in the glycosylation of these proteins were observed. Overall, our work points toward a common mechanism for bacterially induced sepsis, marked by conserved changes in the glycome.

关键词： serum glycomics core 1 3 O-glycans lectin microarray sepsis interalpha-trypsin inhibitor heavy chains (ITIHs) fibronectin

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Effectiveness of Selenium on Chondrocyte Glycoprotein Glycosylation Which Play Important Roles in the Pathogenesis of an Endemic Osteoarthritis, Kashin-Beck Disease

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BIOLOGICAL TRACE ELEMENT RESEARCH 2022年第4期200卷 1531-1537页

作者： Wang, Sen Geng, Lingling Zhao, Guanghui Meng, Peilin Yuan, Linlin Guo, Xiong Xi An Jiao Tong Univ Sch Publ Hlth Hlth Sci Ctr Key Lab Trace Elements & Endem DisNatl Hlth Comm Xian Shaanxi Peoples R China Xian Childrens Hosp Dept Rheumatol & Immunol Xian Shaanxi Peoples R China Xi An Jiao Tong Univ Xian Honghui Hosp Hlth Sci Ctr Xian Shaanxi Peoples R China

In this study, we aimed to explore the effectiveness of selenium on the chondrocyte glycoprotein glycosylation which plays important roles in the pathogenesis of Kashin-Beck disease (KBD). Cartilage samples were collected from KBD patients after total knee replacement surgery. Chondrocytes were cultured with sodium selenium. The group of chondrocytes which were cultured without adding sodium selenium was considered as control group. lectin microarray was used to screen the differences in lectin levels between KBD and KBD with selenium groups. Stronger signals for Bandeiraea simplicifolia (BS-I), Hippeastrum hybrid lectin (HHL), Pisum sativum agglutinin (PSA), Psophocarpus tetragonolobus lectin I (PTL-I), Psophocarpus tetragonolobus lectin II (PTL-II), Sophora japonica agglutinin (SJA), Lotus tetragonolobus lectin (LTL), and Triticum vulgaris (WGA) were observed in the KBD group. Meanwhile, Aleuria aurantia lectin (AAL), Lens culinaris agglutinin (LCA), Lycopersicon esculentum (tomato) lectin (LEL), Peanut agglutinin (PNA), and Sambucus nigra lectin (SNA) signals were lower in the KBD group. Selenium may have the function of influence the expression levels of carbohydrate chains Gal alpha 1,3-Gal, high mannose, and GlcNAc.

关键词： Kashin-Beck disease Chondrocyte Selenium Carbohydrate chain lectin microarray

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Glycosylation of recombinant adeno-associated virus 6

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MOLECULAR THERAPY METHODS & CLINICAL DEVELOPMENT 2024年第2期32卷 101256页

作者： Yamaguchi, Yuki Ishii, Kentaro Koizumi, Sachiko Sakaue, Hiroaki Maruno, Takahiro Fukuhara, Mitsuko Shibuya, Risa Tsunaka, Yasuo Matsushita, Aoba Bandoh, Karin Torisu, Tetsuo Murata-Kishimoto, Chie Tomioka, Azusa Mizukado, Saho Kaji, Hiroyuki Kashiwakura, Yuji Ohmori, Tsukasa Kuno, Atsushi Uchiyama, Susumu Osaka Univ Grad Sch Engn Dept Biotechnol 2-1 Yamadaoka Suita Osaka 5650871 Japan GlycoTechn Ltd Yokohama Japan Precis Syst Sci Co Ltd 88 Kamihongo Matsudo Chiba 2710064 Japan Natl Inst Adv Ind Sci & Technol Cellular & Mol Biotechnol Res Inst Mol & Cellular Glycoprote Res Grp Tsukuba Ibaraki 3058565 Japan U Medico Inc 2-1 Yamadaoka Suita Osaka 5650871 Japan Nagoya Univ Inst Glycocore Res IGCORE Furo ChoChikusa Nagoya Aichi 4648601 Japan Jichi Med Univ Sch Med Dept Biochem 3111-1 Yakushiji Shimotsuke Tochigi 3290498 Japan Jichi Med Univ Ctr Gene Therapy Res 3111-1 Yakushiji Shimotsuke Tochigi 3290498 Japan

Glycosylation of biopharmaceuticals can affect their safety and efficacy. Glycans can occur on recombinant adeno-associated viruses (rAAVs) that are used for gene therapy;however, the types of glycans that attach to rAAVs are controversial. Here, we conducted lectin microarray analyses on six rAAV serotype 6 (rAAV6) preparations that were produced differently. We demonstrate that O-glycans considered to be attached to rAAV6 were recognized by Agaricus bisporus agglutinin (ABA) and that N-glycans were detected in rAAV6 purified without affinity chromatography. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the N-glycans detected in rAAV6 were derived from host cell proteins. A combination of ABA-based fractionation and LC-MS/ MS revealed that rAAV6 was O-glycosylated with the mucintype glycans, O-GalNAc (Tn antigen), and mono- and di-sialylated Galb1-3GalNAc (T antigen) at S156, T162, T194, and T201 in viral protein (VP) 2 and with O-GlcNAc at T242 in VP3. The mucin-type O-glycosylated rAAV6 particles were 0.1%-1% of total particles. Further physicochemical and biological analyses revealed that mucin-type O-glycosylated rAAV6 had a lower ratio of VP1 to VP2/VP3, resulting in a lower transduction efficiency both in vitro and in vivo compared with rAAV6 without mucin-type O-glycans. This report details conclusive evidence of rAAV glycosylation and its impact on rAAV-based therapeutics.

关键词： gene therapy adeno-associated virus glycosylation lectin microarray liquid chromatography-tandem mass spectrometry transduction efficiency

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