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Chen L, Li Y, Guo Y, Wang G, Feng N, Sun J, Zhong Y, Yao Y, Ding L, Ju H. Two-Level Spatially Localized Proximity Labeling for Cross-Biological-Hierarchy Measurement and Manipulation. Angew Chem Int Ed Engl 2025; 64:e202421448. [PMID: 39805739 DOI: 10.1002/anie.202421448] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2024] [Revised: 12/31/2024] [Accepted: 01/13/2025] [Indexed: 01/16/2025]
Abstract
Proximity labeling (PL) has emerged as a powerful technique for the in situ elucidation of biomolecular interaction networks. However, PL methods generally rely on single-biological-hierarchy control of spatial localization at the labeling site, which limits their application in multi-tiered biological systems. Here, we introduced another enzymatic reaction upstream of an enzyme-based PL reaction and targeted the two enzymes to markers indicating different biological hierarchies, establishing a two-level spatially localized proximity labeling (P2L) platform for in situ molecular measurement and manipulation. Using the cellular- and glycan-level as the hierarchical models, we demonstrated the ability of P2L to efficiently execute a two-step logic operation and to discriminate target cells with different levels of glycosylation within mixed cell populations. By mounting clickable handles via P2L, we reprogrammed the robust covalent assembly of cells at designated sites. The combination of P2L with proteomics led to the profiling of the protein microenvironment of specific glycans on target cells, revealing changes in tumor-cell-surface interactions under immune pressure from a glycan perspective. P2L provides not only a solution for revealing the heterogeneity of biological systems, but also new insights in the fields of intelligent logic computation, enzyme engineering, tissue engineering, etc.
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Affiliation(s)
- Liusheng Chen
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China
| | - Yiran Li
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China
| | - Yuna Guo
- School of Clinical and Basic Medical Sciences, Shandong First Medical University, Jinan, 250117, China
| | - Guyu Wang
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China
| | - Nan Feng
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China
| | - Jiahui Sun
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China
| | - Yihong Zhong
- College of Chemistry and Materials, Jiangxi Normal University, Nanchang, 330022, China
| | - Yunyan Yao
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China
| | - Lin Ding
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China
- Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center, Nanjing University, Nanjing, 210023, China
| | - Huangxian Ju
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China
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2
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Wu Y, You Y, Jiang T, He Y, Fan Q, Zeng X, Li T, Lu Y, Qi L, Zhou F, Sun L, Wang D, Zou Y, Zhang G, Yuan Y, Mao Y. Proximity Labeling and Genetic Screening Reveal that DSG2 is a Counter Receptor of Siglec-9 and Suppresses Macrophage Phagocytosis. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2025; 12:e2406654. [PMID: 39813162 PMCID: PMC11884560 DOI: 10.1002/advs.202406654] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/16/2024] [Revised: 12/23/2024] [Indexed: 01/18/2025]
Abstract
Cancer cells present sialylated glycoconjugates that modulate the activity of various immune cells within the tumor microenvironment through trans interaction with immunosuppressive Siglec receptors. Identifying counter receptors for Siglecs can provide valuable targets for cancer immunotherapy, but it presents significant challenges. Here, the identification of DSG2 (Desmoglein 2) as a dominant counter receptor of Siglec-9 in melanoma cells is reported, using a workflow that combines the strength of proximity labeling and the advantage of CRISPR knockout screening. It is further demonstrated that the interaction between DSG2 and Siglec-9 is mainly dependent on sialic acid-bearing N-glycans on DSG2. Importantly, blocking trans interaction between DSG2 and Siglec-9 significantly enhances macrophage phagocytosis of melanoma cells and, to a lesser extent, other cancer cells. The work thus suggests sialylated DSG2 as a potential "don't eat me" signal molecule with therapeutic potentials in cancer immunotherapy.
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Affiliation(s)
- Ying Wu
- School of Pharmaceutical SciencesSun Yat‐sen UniversityGuangzhou510006China
| | - Yuyu You
- School of Pharmaceutical SciencesSun Yat‐sen UniversityGuangzhou510006China
| | - Tingsong Jiang
- School of Pharmaceutical SciencesSun Yat‐sen UniversityGuangzhou510006China
| | - Yuqi He
- School of Pharmaceutical SciencesSun Yat‐sen UniversityGuangzhou510006China
| | - Qingchi Fan
- School of Pharmaceutical SciencesSun Yat‐sen UniversityGuangzhou510006China
| | - Xinlei Zeng
- School of Pharmaceutical SciencesSun Yat‐sen UniversityGuangzhou510006China
| | - Ting Li
- School of Pharmaceutical SciencesSun Yat‐sen UniversityGuangzhou510006China
| | - Yuxing Lu
- School of Pharmaceutical SciencesSun Yat‐sen UniversityGuangzhou510006China
| | - Liang Qi
- School of Pharmaceutical SciencesSun Yat‐sen UniversityGuangzhou510006China
| | - Fengxia Zhou
- School of Pharmaceutical SciencesSun Yat‐sen UniversityGuangzhou510006China
| | - Lingyu Sun
- School of Pharmaceutical SciencesSun Yat‐sen UniversityGuangzhou510006China
| | - Danyang Wang
- State Key Laboratory of Oncology in South ChinaCollaborative Innovation Center for Cancer MedicineSun Yat‐sen University Cancer CenterGuangzhou510275China
| | - Yong Zou
- Department of Blood TransfusionThe Third Affiliated Hospital of Sun Yat‐sen UniversityGuangzhou510000China
| | - Guigen Zhang
- Institute of Human VirologyDepartment of Pathogen Biology and Biosecurityand Key Laboratory of Tropical Disease Control of Ministry of EducationZhongshan School of MedicineSun Yat‐sen UniversityGuangzhou510080China
| | - Yanqiu Yuan
- School of Pharmaceutical SciencesSun Yat‐sen UniversityGuangzhou510006China
- State Key Laboratory of Anti‐Infective Drug Discovery and DevelopmentSchool of Pharmaceutical SciencesSun Yat‐sen UniversityGuangzhou510006China
| | - Yang Mao
- School of Pharmaceutical SciencesSun Yat‐sen UniversityGuangzhou510006China
- Guangdong Provincial Key Laboratory of Drug Non‐Clinical Evaluation and ResearchGuangzhou510006China
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3
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Fan Y, Song S, Pizzi MP, Zou G, Vykoukal JV, Yoshimura K, Jin J, Calin GA, Waters RE, Gan Q, Wang L, Hanash S, Dhar SS, Ajani JA. Exosomal Galectin-3 promotes peritoneal metastases in gastric adenocarcinoma via microenvironment alterations. iScience 2025; 28:111564. [PMID: 39811647 PMCID: PMC11731617 DOI: 10.1016/j.isci.2024.111564] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2024] [Revised: 06/22/2024] [Accepted: 12/06/2024] [Indexed: 01/16/2025] Open
Abstract
Peritoneal carcinomatosis (PC) in gastric adenocarcinoma (GAC) is the most common metastatic site and leads to a short median survival. Exosomes have been shown to remodel the microenvironment, facilitating tumor metastases. However, the functional component in GAC cell-derived exosomes that remodel the landscape in the peritoneal cavity remains unclear. To address this, we performed in-depth proteomic profiling of ascites-derived exosomes from patients with PC, and we found that Galectin-3 was highly enriched in exosomes derived from malignant ascites. exosomal Galectin-3 was the crucial regulator of PC. Blockage of exosomal Galectin-3 significantly inhibited tumor metastases and prolonged overall survival. Exosomal Galectin-3 activated cancer-associated fibroblasts through integrin α1β1/FAK/Akt/mTOR/CXCL12 signaling. Combined inhibition of the CXCL12-CXCR4 axis and exosomal Galectin-3 enhanced the efficacy of anti-PD-1 immunotherapy, leading to significantly diminished PC progression and durable antitumor responses. These findings provide a rationale for clinical strategy of targeting exosomal Galectin-3 to treat PC.
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Affiliation(s)
- Yibo Fan
- Department of Gastrointestinal Medical Oncology, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Shumei Song
- Coriell Institute for Medical Research, Camden, NJ 08103, USA
| | - Melissa Pool Pizzi
- Department of Gastrointestinal Medical Oncology, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Gengyi Zou
- Department of Gastrointestinal Medical Oncology, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Jody V Vykoukal
- Department of Clinical Cancer Prevention, Division of Cancer Prevention and Population Sciences, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Katsuhiro Yoshimura
- Department of Gastrointestinal Medical Oncology, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
- Department of Tumor Pathology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-3192, Japan
| | - Jiankang Jin
- Department of Gastrointestinal Medical Oncology, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - George A Calin
- Department of Translational Molecular Pathology, Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Rebecca E Waters
- Department of Pathology, Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Qiong Gan
- Department of Pathology, Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Linghua Wang
- Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Samir Hanash
- Department of Clinical Cancer Prevention, Division of Cancer Prevention and Population Sciences, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Shilpa S Dhar
- Department of Gastrointestinal Medical Oncology, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Jaffer A Ajani
- Department of Gastrointestinal Medical Oncology, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
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Critcher M, Pang JM, Huang ML. Mapping the FGF2 Interactome Identifies a Functional Proteoglycan Coreceptor. ACS Chem Biol 2025; 20:105-116. [PMID: 39704408 PMCID: PMC11858877 DOI: 10.1021/acschembio.4c00475] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2024]
Abstract
Fibroblast growth factor 2 (FGF2) is a multipotent growth factor and signaling protein that exhibits broad functions across multiple cell types. These functions are often initiated by binding to growth factor receptors and fine-tuned by glycosaminoglycan (GAG)-modified proteins called proteoglycans. The various outputs of FGF2 signaling and functions arise from a dynamic and cell type-specific set of binding partners. However, the interactome of FGF2 has yet to be comprehensively determined. Moreover, the identity of the proteoglycan proteins carrying GAG chains is often overlooked and remains unknown in most cell contexts. Here, we perform peroxidase-catalyzed live cell proximity labeling using an engineered APEX2-FGF2 fusion protein to map the interactome of FGF2. Across two cell lines with established and distinct FGF2-driven functions, we greatly expand upon the known FGF2 interactome, identifying >600 new putative FGF2 interactors. Notably, our results demonstrate a key role for the GAG binding capacity of FGF2 in modulating its interactome.
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Affiliation(s)
- Meg Critcher
- Department of Chemistry, Scripps Research, 10550 N Torrey Pines Rd, La Jolla, California 92037, United States
- Skaggs Graduate School of Chemical and Biological Sciences, Scripps Research, 10550 N Torrey Pines Rd, La Jolla California 92037, United States
| | - Jia Meng Pang
- Department of Chemistry, Scripps Research, 10550 N Torrey Pines Rd, La Jolla, California 92037, United States
- Skaggs Graduate School of Chemical and Biological Sciences, Scripps Research, 10550 N Torrey Pines Rd, La Jolla California 92037, United States
| | - Mia L Huang
- Department of Chemistry, Scripps Research, 10550 N Torrey Pines Rd, La Jolla, California 92037, United States
- Skaggs Graduate School of Chemical and Biological Sciences, Scripps Research, 10550 N Torrey Pines Rd, La Jolla California 92037, United States
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5
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Tang B, Jin C, Li M, Liu S, Zhang X, Li J, Ding K, Zang Y. A novel pectin-like polysaccharide from Crocus sativus targets Galectin-3 to inhibit hepatic stellate cells activation and liver fibrosis. Carbohydr Polym 2025; 348:122826. [PMID: 39562101 DOI: 10.1016/j.carbpol.2024.122826] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2024] [Revised: 09/26/2024] [Accepted: 10/01/2024] [Indexed: 11/21/2024]
Abstract
Liver fibrosis may lead to cirrhosis and even cancer without effective clinical medicine available. Previous studies demonstrated that galactan-containing pectins or pectin-like polysaccharides might target Galectin-3 (Gal-3) to impede fibrosis. This research aims to discover novel pectin-like galactan to interfere with fibrosis for potential new drug development. Thus, we purify a novel homogeneous Rhamnogalacturonan-I like polysaccharide with galactan input, XHH2, from the Crocus sativus flower. XHH2 (MW: 35.7 kDa) consists of rhamnose, galacturonic acid, galactose, and arabinose in ratios of 6.6: 6.1: 25.2: 12.1. The backbone of XHH2 comprises 1, 3, 6-Gal and 1, 3, 4-GalA, with branches at O-3 of 1, 3, 6-Gal and O-4 of 1, 3, 4-GalA. O-3 branches include 1, 3-Gal, 1, 4-Gal, 1, 6-Gal, T-Gal, and T-Glc, while O-4 branches consist of 1, 2, 4-Rha, 1, 4-GalA, 1, 5-Ara, T-Ara, T-Gal, and T-α-HexA. Surface plasmon resonance measurement shows that XHH2 binds to both Gal-3 and integrin β1 to block Gal-3/integrin β1 interaction. Mechanism studies further suggest that XHH2 inactivates hepatic stellate cells (HSCs) via disturbing the Gal-3/Integrin-β1/FAK pathway to alleviate liver injury and fibrosis in vitro & in vivo. XHH2 shows a favorable drug safety in the acute toxicity test of oral administration of XHH2 in mice. Overall, XHH2 is an active ingredient against liver fibrosis by targeting the interaction between Gal-3 and Integrin-β1.
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Affiliation(s)
- Bixi Tang
- Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China; State Key Laboratory of Chemical Biology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
| | - Can Jin
- State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan Tsuihang New District; Zhongshan, Guangzhou 528400, China
| | - Maoting Li
- Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan Tsuihang New District; Zhongshan, Guangzhou 528400, China; School of Pharmaceutical Sciences, Guizhou Medical University, Guiyang 550025, China
| | - Siqi Liu
- School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China
| | - Xuemei Zhang
- Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China.
| | - Jia Li
- State Key Laboratory of Chemical Biology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; Drug Discovery Shandong Laboratory, Bohai Rim Advanced Research Institute for Drug Discovery, Yantai, Shandong 264117, China; Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan Tsuihang New District; Zhongshan, Guangzhou 528400, China; School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, UCAS, Hangzhou, Zhejiang 310024, China; School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China.
| | - Kan Ding
- State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan Tsuihang New District; Zhongshan, Guangzhou 528400, China.
| | - Yi Zang
- State Key Laboratory of Chemical Biology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; Lingang Laboratory, Shanghai 201203, China.
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Wang B, He X, Zhou Y, Tan Z, Li X, Guan F, Lei L. Proximity Labeling-Based Identification of MGAT3 Substrates and Revelation of the Tumor-Suppressive Role of Bisecting GlcNAc in Breast Cancer via GLA Degradation. Cells 2025; 14:103. [PMID: 39851531 PMCID: PMC11764451 DOI: 10.3390/cells14020103] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2024] [Revised: 12/18/2024] [Accepted: 01/10/2025] [Indexed: 01/26/2025] Open
Abstract
Glycosylation plays a critical role in various biological processes, yet identifying specific glycosyltransferase substrates remains a challenge due to the complexity of glycosylation. Here, we employ proximity labeling with biotin ligases BASU and TurboID to map the proximitome of MGAT3, a glycosyltransferase responsible for the biosynthesis of the bisecting GlcNAc structure, in HEK293T cells. This approach enriched 116 and 189 proteins, respectively, identifying 17 common substrates shared with bisecting GlcNAc-bearing proteome obtained via intact glycopeptide enrichment methods. Gene ontology analysis revealed that the enriched proteins were predominantly localized in the exosome, endoplasmic reticulum, and Golgi apparatus, consistent with subcellular localization of MGAT3 substrates. Notably, four novel substrates, GOLM2, CCDC134, ASPH, and ERO1A, were confirmed to bear bisecting GlcNAc modification, validating the utility of the proximity labeling method. Furthermore, we observed that bisecting GlcNAc modification inhibits breast cancer progression by promoting the degradation of α-galactosidase A (GLA). These findings demonstrate the efficacy of proximity labeling in identifying glycosyltransferase substrates and provide insights into the functional impact of bisecting GlcNAc modification.
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Affiliation(s)
- Bowen Wang
- Key Laboratory of Resource Biology and Biotechnology Western China, Ministry of Education, Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University, Xi’an 710069, China; (B.W.); (Y.Z.)
| | - Xin He
- Department of Functional Laboratory, College of Laboratory Medicine, Dalian Medical University, Dalian 116044, China;
| | - Yue Zhou
- Key Laboratory of Resource Biology and Biotechnology Western China, Ministry of Education, Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University, Xi’an 710069, China; (B.W.); (Y.Z.)
| | - Zengqi Tan
- Institute of Hematology, School of Medicine, Northwest University, Xi’an 710069, China; (Z.T.); (X.L.)
| | - Xiang Li
- Institute of Hematology, School of Medicine, Northwest University, Xi’an 710069, China; (Z.T.); (X.L.)
| | - Feng Guan
- Key Laboratory of Resource Biology and Biotechnology Western China, Ministry of Education, Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University, Xi’an 710069, China; (B.W.); (Y.Z.)
| | - Lei Lei
- Key Laboratory of Resource Biology and Biotechnology Western China, Ministry of Education, Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University, Xi’an 710069, China; (B.W.); (Y.Z.)
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7
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Wang X, Qin G, Yang J, Zhao C, Ren J, Qu X. A subcellular selective APEX2-based proximity labeling used for identifying mitochondrial G-quadruplex DNA binding proteins. Nucleic Acids Res 2025; 53:gkae1259. [PMID: 39718986 PMCID: PMC11724306 DOI: 10.1093/nar/gkae1259] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2024] [Revised: 12/02/2024] [Accepted: 12/10/2024] [Indexed: 12/26/2024] Open
Abstract
G-quadruplexes (G4s), as an important type of non-canonical nucleic acid structure, have received much attention because of their regulations of various biological processes in cells. Identifying G4s-protein interactions is essential for understanding G4s-related biology. However, current strategies for exploring G4 binding proteins (G4BPs) include pull-down assays in cell lysates or photoaffinity labeling, which are lack of sufficient spatial specificity at the subcellular level. Herein, we develop a subcellular selective APEX2-based proximity labeling strategy to investigate the interactome of mitochondrial DNA (mtDNA) G4s in living cells. By this method, we have identified several mtDNA G4BPs. Among them, a previously unrecognized mtDNA G4BP, DHX30 has been selected as an example to explore its important biofunctions. DHX30 localizes both in cytoplasm and mitochondria and can resolve mtDNA G4s. Further studies have demonstrated that DHX30 unfolds mtDNA G4 in living cells, which results in a decrease in glycolysis activity of tumor cells. Besides, RHPS4, a known mtDNA G4 stabilizer, will reverse this inhibition effect. Benefiting from the high spatiotemporal resolution and the ability of genetically encoded systems to perform the labeling with exquisite specificity within living cells, our approach can realize the identification of subcellular localized G4BPs. Our work provides a novel strategy to map protein interactions of specific nucleic acid features in subcellular compartments of living cells.
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Affiliation(s)
- Xu Wang
- Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, P. R. China
- University of Science and Technology of China, 96 JinZhai Road, Hefei, Anhui 230026, P. R. China
| | - Geng Qin
- Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, P. R. China
- University of Science and Technology of China, 96 JinZhai Road, Hefei, Anhui 230026, P. R. China
| | - Jie Yang
- Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, P. R. China
- University of Science and Technology of China, 96 JinZhai Road, Hefei, Anhui 230026, P. R. China
| | - Chuanqi Zhao
- Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, P. R. China
- University of Science and Technology of China, 96 JinZhai Road, Hefei, Anhui 230026, P. R. China
| | - Jinsong Ren
- Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, P. R. China
- University of Science and Technology of China, 96 JinZhai Road, Hefei, Anhui 230026, P. R. China
| | - Xiaogang Qu
- Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, P. R. China
- University of Science and Technology of China, 96 JinZhai Road, Hefei, Anhui 230026, P. R. China
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8
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Williams CA, Stone DJ, Joshi SY, Yilmaz G, Farzeen P, Jeon S, Harris-Ryden Z, Becer CR, Deshmukh SA, Callmann CE. Systematic Evaluation of Macromolecular Carbohydrate-Lectin Recognition Using Precision Glycopolymers. Biomacromolecules 2024; 25:7985-7994. [PMID: 39503854 DOI: 10.1021/acs.biomac.4c01245] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/10/2024]
Abstract
The precise modulation of protein-carbohydrate interactions is critical in glycobiology, where multivalent binding governs key cellular processes. As such, synthetic glycopolymers are useful for probing these interactions. Herein, we developed precision glycopolymers (PGPs) with unambiguous local chemical composition and well-defined global structure and systematically evaluated the effect of polymer length, hydrophobicity, and backbone hybridization as well as glycan density and identity on the binding to both mammalian and plant lectins. Our studies identified glycan density as a critical factor, with PGPs below 50% grafting density showing significantly weaker lectin interactions. Coarse-grained molecular dynamics simulations suggest that the observed phenomena may be due to a decrease in carbohydrate-carbohydrate interactions in fully grafted PGPs, leading to improved solvent accessibility. In functional assays, these PGPs reduced the cell viability and migration in 4T1 breast cancer cells. Our findings establish a structure-activity relationship in glycopolymers, providing new strategies for designing synthetic glycomacromolecules for a myriad of applications.
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Affiliation(s)
- Cole A Williams
- Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States
| | - Daniel J Stone
- Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States
| | - Soumil Y Joshi
- Department of Chemical Engineering, Virginia Tech, Blacksburg, Virginia 24061, United States
| | - Gokhan Yilmaz
- Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K
| | - Parisa Farzeen
- Department of Chemical Engineering, Virginia Tech, Blacksburg, Virginia 24061, United States
| | - Sungjin Jeon
- Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States
| | - Zamira Harris-Ryden
- Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States
| | - C Remzi Becer
- Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K
| | - Sanket A Deshmukh
- Department of Chemical Engineering, Virginia Tech, Blacksburg, Virginia 24061, United States
| | - Cassandra E Callmann
- Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States
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9
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Liu X, Yi L, Lin Z, Chen S, Wang S, Sheng Y, Lebrilla CB, Garcia BA, Xie Y. Metabolic Control of Glycosylation Forms for Establishing Glycan-Dependent Protein Interaction Networks. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.10.30.621210. [PMID: 39554187 PMCID: PMC11565926 DOI: 10.1101/2024.10.30.621210] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/19/2024]
Abstract
Protein-protein interactions (PPIs) provide essential insights into the complex molecular mechanisms and signaling pathways within cells that regulate development and disease-related phenotypes. However, for membrane proteins, the impact of various forms of glycosylation has often been overlooked in PPI studies. In this study, we introduce a novel approach, glycan-dependent affinity purification followed by mass spectrometry (GAP-MS), to assess variations in PPIs for any glycoprotein of interest under different glycosylation conditions. As a proof of principle, we selected four glycoproteins-BSG, CD44, EGFR, and SLC3A2-as baits to compare their co-purified partners across five metabolically controlled glycan conditions. The findings demonstrate the capability of GAP-MS to identify PPIs influenced by altered glycosylation states, establishing a foundation for systematically exploring the Glycan-Dependent Protein Interactome (GDPI) for other glycoproteins of interest.
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Affiliation(s)
- Xingyu Liu
- State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences and Institutes of Biomedical Sciences, Fudan University, Shanghai, China
- Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, United States
| | - Li Yi
- State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences and Institutes of Biomedical Sciences, Fudan University, Shanghai, China
| | - Zongtao Lin
- Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, United States
| | - Siyu Chen
- Department of Chemistry, University of California, Davis, Davis, California, United States
| | - Shunyang Wang
- Department of Chemistry, University of California, Davis, Davis, California, United States
| | - Ying Sheng
- Department of Chemistry, University of California, Davis, Davis, California, United States
| | - Carlito B. Lebrilla
- Department of Chemistry, University of California, Davis, Davis, California, United States
- Department of Biochemistry, University of California, Davis, Davis, California, United States
| | - Benjamin A. Garcia
- Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, United States
| | - Yixuan Xie
- State Key Laboratory of Genetic Engineering, Greater Bay Area Institute of Precision Medicine (Guangzhou), School of Life Sciences and Institutes of Biomedical Sciences, Fudan University, Shanghai, China
- Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, United States
- Department of Chemistry, University of California, Davis, Davis, California, United States
- Lead contact
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10
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Fan N, Yang K, Bian X, Chen Y, Zhang L, Ai Z, Li X, Ding S, Li S, Cheng W. GlycoSS: A DNA Glycosignal Sieve for Deciphering Spatially Resolved EpCAM-Specific Glycoforms. ACS NANO 2024; 18:29106-29120. [PMID: 39374425 DOI: 10.1021/acsnano.4c10565] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/09/2024]
Abstract
Malignant transformation of cancer is often accompanied by aberrant glycopatterns. Epithelial-mesenchymal transition (EMT) is a crucial biological process in cancer migration and invasion, accelerating cancer deterioration. High-precision analysis of protein-glycan spatial profiling in the EMT process is essential for elucidating glycosylation functions and cancer progression. However, the diversity of glycans in composition and conformation complicates their spatial analysis. Here, we develop a DNA glycosignal sieve (GlycoSS) visualization platform for screening glycoform expression with a protein spatial dimension. GlycoSS utilizes protein-anchored DNA nanoscanners of distinct lengths to control glycosignal readout, enabling protein-glycan distance modulations, and simultaneously orthogonally amplify glycoform output through signal amplification by an exchange reaction. Using GlycoSS, we screened EpCAM-specific hypoglycosylated glycoform signals in different breast cancer cell subtypes, especially characterizing the spatial distribution of glycans on the MCF-7 cell surface. Considering that the EpCAM-specific N-glycan dysregulation in EMT is pivotal, GlycoSS revealed dynamic glycan fluctuations during IGF-1-induced EMT, revealing that the N-glycans were positively associated with tumor malignancy and metastasis. GlycoSS is anticipated to accelerate the identification of aberrant N-glycosylation in tumor progression, advancing systemic glycobiology insights. Notably, GlycoSS is capable of analyzing diverse glycoprotein profiles, offering additional dimensions into the role of glycoprotein nanoenvironments in regulating membrane protein function.
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Affiliation(s)
- Ningke Fan
- Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
| | - Ketong Yang
- Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
| | - Xintong Bian
- Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
| | - Yirong Chen
- Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
| | - Lu Zhang
- Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
| | - Zhujun Ai
- Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
| | - Xinyu Li
- Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
| | - Shijia Ding
- Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
| | - Siqiao Li
- School of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016, China
| | - Wei Cheng
- The Center for Clinical Molecular Medical Detection, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
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11
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Calver JF, Parmar NR, Harris G, Lithgo RM, Stylianou P, Zetterberg FR, Gooptu B, Mackinnon AC, Carr SB, Borthwick LA, Scott DJ, Stewart ID, Slack RJ, Jenkins RG, John AE. Defining the mechanism of galectin-3-mediated TGF-β1 activation and its role in lung fibrosis. J Biol Chem 2024; 300:107300. [PMID: 38641066 PMCID: PMC11134550 DOI: 10.1016/j.jbc.2024.107300] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2023] [Revised: 03/29/2024] [Accepted: 04/11/2024] [Indexed: 04/21/2024] Open
Abstract
Integrin-mediated activation of the profibrotic mediator transforming growth factor-β1 (TGF-β1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 is believed to contribute to the pathological wound healing seen in IPF, although its mechanism of action is not precisely defined. We hypothesized that galectin-3 potentiates TGF-β1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-β1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L-α-lysophosphatidic acid sodium salt-induced integrin-mediated TGF-β1 activation. Surface plasmon resonance analysis confirmed that galectin-3 binds to αv integrins, αvβ1, αvβ5, and αvβ6, and to the TGFβRII subunit in a glycosylation-dependent manner. This binding is heterogeneous and not a 1:1 binding stoichiometry. Binding interactions were blocked by small molecule inhibitors of galectin-3, which target the carbohydrate recognition domain. Galectin-3 binding to β1 integrin was validated in vitro by coimmunoprecipitation in HLFs. Proximity ligation assays indicated that galectin-3 and β1 integrin colocalize closely (≤40 nm) on the cell surface and that colocalization is increased by TGF-β1 treatment and blocked by galectin-3 inhibitors. In the absence of TGF-β1 stimulation, colocalization was detectable only in HLFs from IPF patients, suggesting the proteins are inherently more closely associated in the disease state. Galectin-3 inhibitor treatment of precision cut lung slices from IPF patients' reduced Col1a1, TIMP1, and hyaluronan secretion to a similar degree as TGF-β type I receptor inhibitor. These data suggest that galectin-3 promotes TGF-β1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-β1 signaling cascade.
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Affiliation(s)
- Jessica F Calver
- School of Medicine, University of Nottingham, Nottingham, United Kingdom; Stevenage Bioscience Catalyst, Galecto Biotech AB, Stevenage, United Kingdom
| | - Nimesh R Parmar
- School of Medicine, University of Nottingham, Nottingham, United Kingdom; Roche Products Limited, Welwyn Garden City, Hertfordshire, United Kingdom
| | - Gemma Harris
- Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, Oxfordshire, United Kingdom
| | - Ryan M Lithgo
- Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, Oxfordshire, United Kingdom; School of Biosciences, University of Nottingham, Loughborough, Leicestershire, United Kingdom; Membrane Protein Laboratory, Diamond Light Source, Rutherford Appleton Laboratory, Didcot, Oxfordshire, United Kingdom; Diamond Light Source, Diamond House, Rutherford Appleton Laboratories, Didcot, Oxfordshire, United Kingdom
| | - Panayiota Stylianou
- Institute for Lung Health, NIHR Leicester Respiratory Biomedical Research Centre, Glenfield Hospital, Leicester, United Kingdom; Leicester Institute for Structural and Chemical Biology, Henry Wellcome Building, University of Leicester, Leicester, United Kingdom
| | | | - Bibek Gooptu
- Institute for Lung Health, NIHR Leicester Respiratory Biomedical Research Centre, Glenfield Hospital, Leicester, United Kingdom; Leicester Institute for Structural and Chemical Biology, Henry Wellcome Building, University of Leicester, Leicester, United Kingdom
| | - Alison C Mackinnon
- Galecto Biotech AB, Nine Edinburgh BioQuarter, Edinburgh, United Kingdom
| | - Stephen B Carr
- Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, Oxfordshire, United Kingdom; Department of Chemistry, University of Oxford, Oxford, Oxfordshire, United Kingdom
| | - Lee A Borthwick
- Fibrofind Ltd, Newcastle upon Tyne, United Kingdom; Newcastle Fibrosis Research Group, Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom
| | - David J Scott
- Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, Oxfordshire, United Kingdom; School of Biosciences, University of Nottingham, Loughborough, Leicestershire, United Kingdom
| | - Iain D Stewart
- National Heart and Lung Institute, Imperial College London, London, United Kingdom
| | - Robert J Slack
- Stevenage Bioscience Catalyst, Galecto Biotech AB, Stevenage, United Kingdom
| | - R Gisli Jenkins
- National Heart and Lung Institute, Imperial College London, London, United Kingdom
| | - Alison E John
- National Heart and Lung Institute, Imperial College London, London, United Kingdom.
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12
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Nelson ZM, Kadiri O, Fehl C. GlycoID Proximity Labeling to Identify O-GlcNAcylated Protein Interactomes in Live Cells. Curr Protoc 2024; 4:e1052. [PMID: 38752278 PMCID: PMC11101149 DOI: 10.1002/cpz1.1052] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/19/2024]
Abstract
Cells continuously remodel their intracellular proteins with the monosaccharide O-linked N-acetylglucosamine (O-GlcNAc) to regulate metabolism, signaling, and stress. This protocol describes the use of GlycoID tools to capture O-GlcNAc dynamics in live cells. GlycoID constructs contain an O-GlcNAc binding domain linked to a proximity labeling domain and a subcellular localization sequence. When expressed in mammalian cells, GlycoID tracks changes in O-GlcNAc-modified proteins and their interactomes in response to chemical induction with biotin over time. Pairing the subcellular localization of GlycoID with the chemical induction of activity enables spatiotemporal studies of O-GlcNAc biology during cellular events such as insulin signaling. However, optimizing intracellular labeling experiments requires attention to several variables. Here, we describe two protocols to adapt GlycoID methods to a cell line and biological process of interest. Next, we describe how to conduct a semiquantitative proteomic analysis of O-GlcNAcylated proteins and their interactomes using insulin versus glucagon signaling as a sample application. This articles aims to establish baseline GlycoID protocols for new users and set the stage for widespread use over diverse cellular applications for the functional study of O-GlcNAc glycobiology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Expression of targeted GlycoID constructs to verify subcellular location and labeling activity in mammalian cells Basic Protocol 2: GlycoID labeling in live HeLa cells for O-GlcNAc proteomic comparisons.
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Affiliation(s)
- Zachary M. Nelson
- Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI, United States
| | - Oseni Kadiri
- Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI, United States
| | - Charlie Fehl
- Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI, United States
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13
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Čaval T, Xu G, Baniasad M, Chu CW, Rice R, Hundal I, Czerwieniec G, Schwarz F. Mass Spectrometry Analysis of Glycopeptides Enriched by Anion Exchange-Mediated Methods Reveals PolyLacNAc-Extended N-Glycans in Integrins and Tetraspanins in Melanoma Cells. Anal Chem 2024; 96:5086-5094. [PMID: 38513651 PMCID: PMC10993200 DOI: 10.1021/acs.analchem.3c04045] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2023] [Revised: 03/05/2024] [Accepted: 03/06/2024] [Indexed: 03/23/2024]
Abstract
Glycosylation is a key modulator of the functional state of proteins. Recent developments in large-scale analysis of intact glycopeptides have enabled the identification of numerous glycan structures that are relevant in pathophysiological processes. However, one motif found in N-glycans, poly-N-acetyllactosamine (polyLacNAc), still poses a substantial challenge to mass spectrometry-based glycoproteomic analysis due to its relatively low abundance and large size. In this work, we developed approaches for the systematic mapping of polyLacNAc-elongated N-glycans in melanoma cells. We first evaluated five anion exchange-based matrices for enriching intact glycopeptides and selected two materials that provided better overall enrichment efficiency. We then tested the robustness of the methodology by quantifying polyLacNAc-containing glycopeptides as well as changes in protein fucosylation and sialylation. Finally, we applied the optimal enrichment methods to discover glycopeptides containing polyLacNAc motifs in melanoma cells and found that integrins and tetraspanins are substantially modified with these structures. This study demonstrates the feasibility of glycoproteomic approaches for identification of glycoproteins with polyLacNAc motifs.
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Affiliation(s)
| | | | - Maryam Baniasad
- InterVenn Biosciences, 2 Tower Place Fifth Floor, South San Francisco, California 94080, United States
| | - Chih-Wei Chu
- InterVenn Biosciences, 2 Tower Place Fifth Floor, South San Francisco, California 94080, United States
| | - Rachel Rice
- InterVenn Biosciences, 2 Tower Place Fifth Floor, South San Francisco, California 94080, United States
| | - Itati Hundal
- InterVenn Biosciences, 2 Tower Place Fifth Floor, South San Francisco, California 94080, United States
| | - Gregg Czerwieniec
- InterVenn Biosciences, 2 Tower Place Fifth Floor, South San Francisco, California 94080, United States
| | - Flavio Schwarz
- InterVenn Biosciences, 2 Tower Place Fifth Floor, South San Francisco, California 94080, United States
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14
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Huang Y, Zhai G, Fu Y, Li Y, Zang Y, Lin Y, Zhang K. A proximity labeling-based orthogonal trap strategy identifies HDAC8 promotes cell motility by modulating cortactin acetylation. Cell Chem Biol 2024; 31:514-522.e4. [PMID: 38460516 DOI: 10.1016/j.chembiol.2024.02.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2023] [Revised: 12/14/2023] [Accepted: 02/16/2024] [Indexed: 03/11/2024]
Abstract
It is a challenge for the traditional affinity methods to capture transient interactions of enzyme-post-translational modification (PTM) substrates in vivo. Herein we presented a strategy termed proximity labeling-based orthogonal trap approach (ProLORT), relying upon APEX2-catalysed proximity labeling and an orthogonal trap pipeline as well as quantitative proteomics to directly investigate the transient interactome of enzyme-PTM substrates in living cells. As a proof of concept, ProLORT allows for robust evaluation of a known HDAC8 substrate, histone H3K9ac. By leveraging this approach, we identified numerous of putative acetylated proteins targeted by HDAC8, and further confirmed CTTN as a bona fide substrate in vivo. Next, we demonstrated that HDAC8 facilitates cell motility via deacetylation of CTTN at lysine 144 that attenuates its interaction with F-actin, expanding the underlying regulatory mechanisms of HDAC8. We developed a general strategy to profile the transient enzyme-substrate interactions mediated by PTMs, providing a powerful tool for identifying the spatiotemporal PTM-network regulated by enzymes in living cells.
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Affiliation(s)
- Yepei Huang
- The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Tianjin Key Laboratory of Medical Epigenetics, Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin 300070, China; Laboratory of Biochemistry and Molecular Biology Research, Department of Clinical Laboratory, Fujian Medical University Cancer Hospital, No. 420 Fuma Road, Jin'an District, Fuzhou 350014, Fujian Province, China
| | - Guijin Zhai
- The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Tianjin Key Laboratory of Medical Epigenetics, Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin 300070, China.
| | - Yun Fu
- Fujian Provincial Sperm bank, Fujian Maternity and Child Health Hospital College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics, Fujian Medical University, Fuzhou 350005, Fujian Province, China
| | - Yanan Li
- The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Tianjin Key Laboratory of Medical Epigenetics, Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin 300070, China
| | - Yong Zang
- The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Tianjin Key Laboratory of Medical Epigenetics, Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin 300070, China
| | - Yu Lin
- Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI 48109, United States.
| | - Kai Zhang
- The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Tianjin Key Laboratory of Medical Epigenetics, Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin 300070, China.
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15
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Milione RR, Schell BB, Douglas CJ, Seath CP. Creative approaches using proximity labeling to gain new biological insights. Trends Biochem Sci 2024; 49:224-235. [PMID: 38160064 PMCID: PMC10939868 DOI: 10.1016/j.tibs.2023.12.005] [Citation(s) in RCA: 8] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2023] [Revised: 12/04/2023] [Accepted: 12/11/2023] [Indexed: 01/03/2024]
Abstract
At its most fundamental level, life is a collection of synchronized cellular processes driven by interactions among biomolecules. Proximity labeling has emerged as a powerful technique to capture these interactions in native settings, revealing previously unexplored elements of biology. This review highlights recent developments in proximity labeling, focusing on methods that push the fundamental technologies beyond the classic bait-prey paradigm, such as RNA-protein interactions, ligand/small-molecule-protein interactions, cell surface protein interactions, and subcellular protein trafficking. The advancement of proximity labeling methods to address different biological problems will accelerate our understanding of the complex biological systems that make up life.
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Affiliation(s)
- Ryan R Milione
- Skaggs Graduate School of Chemical and Biological Sciences, 120 Scripps Way, Jupiter, FL 33458, USA; Department of Chemistry, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, 120 Scripps Way, Jupiter, FL 33458, USA
| | - Bin-Bin Schell
- Skaggs Graduate School of Chemical and Biological Sciences, 120 Scripps Way, Jupiter, FL 33458, USA; Department of Chemistry, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, 120 Scripps Way, Jupiter, FL 33458, USA
| | - Cameron J Douglas
- Skaggs Graduate School of Chemical and Biological Sciences, 120 Scripps Way, Jupiter, FL 33458, USA; Department of Chemistry, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, 120 Scripps Way, Jupiter, FL 33458, USA
| | - Ciaran P Seath
- Skaggs Graduate School of Chemical and Biological Sciences, 120 Scripps Way, Jupiter, FL 33458, USA; Department of Chemistry, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, 120 Scripps Way, Jupiter, FL 33458, USA.
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16
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Querol Cano L, Dunlock VME, Schwerdtfeger F, van Spriel AB. Membrane organization by tetraspanins and galectins shapes lymphocyte function. Nat Rev Immunol 2024; 24:193-212. [PMID: 37758850 DOI: 10.1038/s41577-023-00935-0] [Citation(s) in RCA: 8] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/15/2023] [Indexed: 09/29/2023]
Abstract
Immune receptors are not randomly distributed at the plasma membrane of lymphocytes but are segregated into specialized domains that function as platforms to initiate signalling, as exemplified by the B cell or T cell receptor complex and the immunological synapse. 'Membrane-organizing proteins' and, in particular, tetraspanins and galectins, are crucial for controlling the spatiotemporal organization of immune receptors and other signalling proteins. Deficiencies in specific tetraspanins and galectins result in impaired immune synapse formation, lymphocyte proliferation, antibody production and migration, which can lead to impaired immunity, tumour development and autoimmunity. In contrast to conventional ligand-receptor interactions, membrane organizers interact in cis (on the same cell) and modulate receptor clustering, receptor dynamics and intracellular signalling. New findings have uncovered their complex and dynamic nature, revealing shared binding partners and collaborative activity in determining the composition of membrane domains. Therefore, immune receptors should not be envisaged as independent entities and instead should be studied in the context of their spatial organization in the lymphocyte membrane. We advocate for a novel approach to study lymphocyte function by globally analysing the role of membrane organizers in the assembly of different membrane complexes and discuss opportunities to develop therapeutic approaches that act via the modulation of membrane organization.
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Affiliation(s)
- Laia Querol Cano
- Department of Medical BioSciences, Radboud University Medical Center, Nijmegen, The Netherlands
| | - Vera-Marie E Dunlock
- Department of Medical BioSciences, Radboud University Medical Center, Nijmegen, The Netherlands
- Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Fabian Schwerdtfeger
- Department of Medical BioSciences, Radboud University Medical Center, Nijmegen, The Netherlands
| | - Annemiek B van Spriel
- Department of Medical BioSciences, Radboud University Medical Center, Nijmegen, The Netherlands.
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17
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Stewart N, Daly J, Drummond-Guy O, Krishnamoorthy V, Stark JC, Riley NM, Williams KC, Bertozzi CR, Wisnovsky S. The glycoimmune checkpoint receptor Siglec-7 interacts with T-cell ligands and regulates T-cell activation. J Biol Chem 2024; 300:105579. [PMID: 38141764 PMCID: PMC10831161 DOI: 10.1016/j.jbc.2023.105579] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2023] [Accepted: 11/28/2023] [Indexed: 12/25/2023] Open
Abstract
Siglec-7 (sialic acid-binding immunoglobulin-like lectin 7) is a glycan-binding immune receptor that is emerging as a significant target of interest for cancer immunotherapy. The physiological ligands that bind Siglec-7, however, remain incompletely defined. In this study, we characterized the expression of Siglec-7 ligands on peripheral immune cell subsets and assessed whether Siglec-7 functionally regulates interactions between immune cells. We found that disialyl core 1 O-glycans are the major immune ligands for Siglec-7 and that these ligands are particularly highly expressed on naïve T-cells. Densely glycosylated sialomucins are the primary carriers of these glycans, in particular a glycoform of the cell-surface marker CD43. Biosynthesis of Siglec-7-binding glycans is dynamically controlled on different immune cell subsets through a genetic circuit involving the glycosyltransferase GCNT1. Siglec-7 blockade was found to increase activation of both primary T-cells and antigen-presenting dendritic cells in vitro, indicating that Siglec-7 binds T-cell glycans to regulate intraimmune signaling. Finally, we present evidence that Siglec-7 directly activates signaling pathways in T-cells, suggesting a new biological function for this receptor. These studies conclusively demonstrate the existence of a novel Siglec-7-mediated signaling axis that physiologically regulates T-cell activity. Going forward, our findings have significant implications for the design and implementation of therapies targeting immunoregulatory Siglec receptors.
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Affiliation(s)
- Natalie Stewart
- Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada
| | - John Daly
- Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada
| | - Olivia Drummond-Guy
- Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada
| | - Vignesh Krishnamoorthy
- Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada
| | - Jessica C Stark
- Department of Chemistry & Sarafan ChEM-H, Stanford University, Stanford, California, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Boston, Massachusetts, USA; Department of Chemical Engineering, Massachusetts Institute of Technology, Boston, Massachusetts, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Boston, Massachusetts, USA
| | - Nicholas M Riley
- Department of Chemistry & Sarafan ChEM-H, Stanford University, Stanford, California, USA; Department of Chemistry, University of Washington, Seattle, Washington, USA
| | - Karla C Williams
- Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada
| | - Carolyn R Bertozzi
- Department of Chemistry & Sarafan ChEM-H, Stanford University, Stanford, California, USA; Howard Hughes Medical Institute, Stanford, California, USA
| | - Simon Wisnovsky
- Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
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18
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Ortega-Ferreira C, Soret P, Robin G, Speca S, Hubert S, Le Gall M, Desvaux E, Jendoubi M, Saint-Paul J, Chadli L, Chomel A, Berger S, Nony E, Neau B, Fould B, Licznar A, Levasseur F, Guerrier T, Elouej S, Courtade-Gaïani S, Provost N, Nguyen TQ, Verdier J, Launay D, De Ceuninck F. Antibody-mediated neutralization of galectin-3 as a strategy for the treatment of systemic sclerosis. Nat Commun 2023; 14:5291. [PMID: 37652913 PMCID: PMC10471779 DOI: 10.1038/s41467-023-41117-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2022] [Accepted: 08/22/2023] [Indexed: 09/02/2023] Open
Abstract
Systemic sclerosis (SSc) is an autoimmune, inflammatory and fibrotic disease with limited treatment options. Developing new therapies is therefore crucial to address patient needs. To this end, we focused on galectin-3 (Gal-3), a lectin known to be associated with several pathological processes seen in SSc. Using RNA sequencing of whole-blood samples in a cross-sectional cohort of 249 patients with SSc, Gal-3 and its interactants defined a strong transcriptomic fingerprint associated with disease severity, pulmonary and cardiac malfunctions, neutrophilia and lymphopenia. We developed new Gal-3 neutralizing monoclonal antibodies (mAb), which were then evaluated in a mouse model of hypochlorous acid (HOCl)-induced SSc. We show that two of these antibodies, D11 and E07, reduced pathological skin thickening, lung and skin collagen deposition, pulmonary macrophage content, and plasma interleukin-5 and -6 levels. Moreover, E07 changed the transcriptional profiles of HOCl-treated mice, resulting in a gene expression pattern that resembled that of control mice. Similarly, pathological pathways engaged in patients with SSc were counteracted by E07 in mice. Collectively, these findings demonstrate the translational potential of Gal-3 blockade as a therapeutic option for SSc.
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Affiliation(s)
- Céline Ortega-Ferreira
- Servier R&D Center, Biomarker Assay Development, Translational Medicine, Gif-sur-Yvette, France
| | - Perrine Soret
- Servier R&D Center, Biomarker Biostatistics, Gif-sur-Yvette, France
| | | | - Silvia Speca
- U1286 INFINITE, Institute for Translational Research in Inflammation, Lille University, Gif-sur-Yvette, France
- Inserm, Lille, France
| | - Sandra Hubert
- Servier R&D Center, Neurosciences and Immuno-inflammation Therapeutic Area, Gif-sur-Yvette, France
| | | | - Emiko Desvaux
- Servier R&D Center, Neurosciences and Immuno-inflammation Therapeutic Area, Gif-sur-Yvette, France
| | - Manel Jendoubi
- U1286 INFINITE, Institute for Translational Research in Inflammation, Lille University, Gif-sur-Yvette, France
- Inserm, Lille, France
| | | | - Loubna Chadli
- Servier R&D Center, Clinical Biomarker Development, Translational Medicine, Gif-sur-Yvette, France
| | - Agnès Chomel
- Servier R&D Center, Protein Sciences, Gif-sur-Yvette, France
| | - Sylvie Berger
- Servier R&D Center, Structural Sciences, Gif-sur-Yvette, France
| | - Emmanuel Nony
- Servier R&D Center, Protein Sciences, Gif-sur-Yvette, France
| | - Béatrice Neau
- Servier R&D Center, Preclinical Biostatistics, Quantitative Pharmacology, Gif-sur-Yvette, France
| | - Benjamin Fould
- Servier R&D Center, Protein Sciences, Gif-sur-Yvette, France
| | - Anne Licznar
- Servier R&D Center, DMPK Department, Translational Medicine, Gif-sur-Yvette, France
| | - Franck Levasseur
- Servier R&D Center, DMPK Department, Translational Medicine, Gif-sur-Yvette, France
| | - Thomas Guerrier
- U1286 INFINITE, Institute for Translational Research in Inflammation, Lille University, Gif-sur-Yvette, France
- Inserm, Lille, France
| | - Sahar Elouej
- Servier R&D Center, Computational Medicine, Gif-sur-Yvette, France
| | | | - Nicolas Provost
- Servier R&D Center, Molecular Genomics, Gif-sur-Yvette, France
| | | | - Julien Verdier
- Servier R&D Center, Neurosciences and Immuno-inflammation Therapeutic Area, Gif-sur-Yvette, France
| | - David Launay
- U1286 INFINITE, Institute for Translational Research in Inflammation, Lille University, Gif-sur-Yvette, France
- Inserm, Lille, France
- Lille University Hospital, Department of Internal Medicine and Clinical Immunology, Reference Center for Rare Systemic Autoimmune Diseases, North and North-West France (CeRAINO), Lille, France
| | - Frédéric De Ceuninck
- Servier R&D Center, Neurosciences and Immuno-inflammation Therapeutic Area, Gif-sur-Yvette, France.
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19
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Čaval T, Alisson-Silva F, Schwarz F. Roles of glycosylation at the cancer cell surface: opportunities for large scale glycoproteomics. Theranostics 2023; 13:2605-2615. [PMID: 37215580 PMCID: PMC10196828 DOI: 10.7150/thno.81760] [Citation(s) in RCA: 28] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2022] [Accepted: 04/13/2023] [Indexed: 05/24/2023] Open
Abstract
Cell surface glycosylation has a variety of functions, and its dysregulation in cancer contributes to impaired signaling, metastasis and the evasion of the immune responses. Recently, a number of glycosyltransferases that lead to altered glycosylation have been linked to reduced anti-tumor immune responses: B3GNT3, which is implicated in PD-L1 glycosylation in triple negative breast cancer, FUT8, through fucosylation of B7H3, and B3GNT2, which confers cancer resistance to T cell cytotoxicity. Given the increased appreciation of the relevance of protein glycosylation, there is a critical need for the development of methods that allow for an unbiased interrogation of cell surface glycosylation status. Here we provide an overview of the broad changes in glycosylation at the surface of cancer cell and describe selected examples of receptors with aberrant glycosylation leading to functional changes, with emphasis on immune checkpoint inhibitors, growth-promoting and growth-arresting receptors. Finally, we posit that the field of glycoproteomics has matured to an extent where large-scale profiling of intact glycopeptides from the cell surface is feasible and is poised for discovery of new actionable targets against cancer.
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20
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Zhu L, Tang Y, Li XY, Kerk SA, Lyssiotis CA, Sun X, Wang Z, Cho JS, Ma J, Weiss SJ. Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption. J Cell Biol 2023; 222:e202206121. [PMID: 36880731 PMCID: PMC9998966 DOI: 10.1083/jcb.202206121] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2022] [Revised: 12/18/2022] [Accepted: 01/23/2023] [Indexed: 03/08/2023] Open
Abstract
Bone-resorbing osteoclasts mobilize proteolytic enzymes belonging to the matrix metalloproteinase (MMP) family to directly degrade type I collagen, the dominant extracellular matrix component of skeletal tissues. While searching for additional MMP substrates critical to bone resorption, Mmp9/Mmp14 double-knockout (DKO) osteoclasts-as well as MMP-inhibited human osteoclasts-unexpectedly display major changes in transcriptional programs in tandem with compromised RhoA activation, sealing zone formation and bone resorption. Further study revealed that osteoclast function is dependent on the ability of Mmp9 and Mmp14 to cooperatively proteolyze the β-galactoside-binding lectin, galectin-3, on the cell surface. Mass spectrometry identified the galectin-3 receptor as low-density lipoprotein-related protein-1 (Lrp1), whose targeting in DKO osteoclasts fully rescues RhoA activation, sealing zone formation and bone resorption. Together, these findings identify a previously unrecognized galectin-3/Lrp1 axis whose proteolytic regulation controls both the transcriptional programs and the intracellular signaling cascades critical to mouse as well as human osteoclast function.
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Affiliation(s)
- Lingxin Zhu
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China
- Division of Genetic Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
- Life Sciences Institute, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
| | - Yi Tang
- Division of Genetic Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
- Life Sciences Institute, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
| | - Xiao-Yan Li
- Division of Genetic Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
- Life Sciences Institute, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
| | - Samuel A. Kerk
- Department of Internal Medicine, Division of Gastroenterology, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
- Doctoral Program in Cancer Biology, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
| | - Costas A. Lyssiotis
- Department of Internal Medicine, Division of Gastroenterology, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
- Rogel Cancer Center, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
| | - Xiaoyue Sun
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China
| | - Zijun Wang
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China
| | - Jung-Sun Cho
- Division of Genetic Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
- Life Sciences Institute, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
| | - Jun Ma
- Division of Genetic Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
- Life Sciences Institute, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
| | - Stephen J. Weiss
- Division of Genetic Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
- Life Sciences Institute, University of Michigan, Ann Arbor, Ann Arbor, MI, USA
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21
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Guo Y, Wang N, Zhong Y, Li W, Li Y, Wang G, Yao Y, Shi Y, Chen L, Wang X, Ding L, Ju H. Cell-Selective Multifunctional Surface Covalent Reconfiguration Using Aptamer-Enabled Proximity Catalytic Labeling. J Am Chem Soc 2023; 145:5092-5104. [PMID: 36821097 DOI: 10.1021/jacs.2c11150] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/24/2023]
Abstract
Cell surface engineering provides access to custom-made cell interfaces with desirable properties and functions. However, cell-selective covalent labeling methods that can simultaneously install multiple molecules with different functions are scarce. Herein, we report an aptamer-enabled proximity catalytic covalent labeling platform for multifunctional surface reconfiguration of target cells in mixed cell populations. By conjugating peroxidase with cell-selective aptamers, the probes formed can selectively bind target cells and catalyze target-cell-localized covalent labeling in situ. The universal applicability of the platform to different phenol-modified functional molecules allows us to perform a variety of manipulations on target cells, including labeling, tracking, assembly regulation, and surface remodeling. In particular, the platform has the ability of multiplexed covalent labeling, which can be used to install two mutually orthogonal click reactive molecules simultaneously on the surface of target cells. We thus achieve "multitasking" in complex multicellular systems: programming and tracking specific cell-cell interactions. We further extend the functional molecules to carbohydrates and perform ultrafast neoglycosylation on target living cells. These newly introduced sugars on the cell membrane can be recognized and remodeled by a glycan-modifying enzyme, thus providing a method package for cell-selective engineering of the glycocalyx.
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Affiliation(s)
- Yuna Guo
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.,Medical Science and Technology Innovation Center, Shandong First Medical University, Jinan 250117, China
| | - Nan Wang
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
| | - Yihong Zhong
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
| | - Wei Li
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
| | - Yiran Li
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
| | - Guyu Wang
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
| | - Yunyan Yao
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
| | - Yue Shi
- State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
| | - Liusheng Chen
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
| | - Xiaojian Wang
- Institute of Advanced Synthesis, School of Chemistry and Molecular Engineering, Nanjing Tech University, Nanjing 211816, China
| | - Lin Ding
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.,Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing 210023, China
| | - Huangxian Ju
- State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
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22
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Sun F, Suttapitugsakul S, Wu R. Systematic characterization of extracellular glycoproteins using mass spectrometry. MASS SPECTROMETRY REVIEWS 2023; 42:519-545. [PMID: 34047389 PMCID: PMC8627532 DOI: 10.1002/mas.21708] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/30/2021] [Revised: 05/10/2021] [Accepted: 05/13/2021] [Indexed: 05/13/2023]
Abstract
Surface and secreted glycoproteins are essential to cells and regulate many extracellular events. Because of the diversity of glycans, the low abundance of many glycoproteins, and the complexity of biological samples, a system-wide investigation of extracellular glycoproteins is a daunting task. With the development of modern mass spectrometry (MS)-based proteomics, comprehensive analysis of different protein modifications including glycosylation has advanced dramatically. This review focuses on the investigation of extracellular glycoproteins using MS-based proteomics. We first discuss the methods for selectively enriching surface glycoproteins and investigating protein interactions on the cell surface, followed by the application of MS-based proteomics for surface glycoprotein dynamics analysis and biomarker discovery. We then summarize the methods to comprehensively study secreted glycoproteins by integrating various enrichment approaches with MS-based proteomics and their applications for global analysis of secreted glycoproteins in different biological samples. Collectively, MS significantly expands our knowledge of extracellular glycoproteins and enables us to identify extracellular glycoproteins as potential biomarkers for disease detection and drug targets for disease treatment.
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Affiliation(s)
| | | | - Ronghu Wu
- School of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, USA
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23
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Kram M. Galectin-3 inhibition as a potential therapeutic target in non-alcoholic steatohepatitis liver fibrosis. World J Hepatol 2023; 15:201-207. [PMID: 36926236 PMCID: PMC10011901 DOI: 10.4254/wjh.v15.i2.201] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/06/2022] [Revised: 12/17/2022] [Accepted: 02/08/2023] [Indexed: 02/24/2023] Open
Abstract
Nonalcoholic fatty liver disease continues to be one of the major health challenges facing the world, with estimates of non-alcoholic steatohepatitis (NASH) prevalence in over 25 percent of the world’s population. NASH represents a spectrum of disease that may lead to hepatic fibrosis and eventual cirrhosis, with the risk of cirrhosis decompensation, and hepatocellular carcinoma. New therapies are desperately needed for NASH, especially for later stages of fibrosis and cirrhosis. Galectin-3 inhibition is being explored as a new liver antifibrotic therapy. This concise review will outline the state of the art of this new therapeutic target.
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Affiliation(s)
- Michael Kram
- Department of Gastroenterology, Bon Secours Health System Inc, Monsey, NY 10952, United States
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24
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Ezhilarasan D. Unraveling the pathophysiologic role of galectin-3 in chronically injured liver. J Cell Physiol 2023; 238:673-686. [PMID: 36745560 DOI: 10.1002/jcp.30956] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2022] [Revised: 01/04/2023] [Accepted: 01/12/2023] [Indexed: 02/07/2023]
Abstract
Galectin-3 (Gal-3) previously referred to as S-type lectins, is a soluble protein that specifically binds to β-galactoside carbohydrates with high specificity. Gal-3 plays a pivotal role in a variety of pathophysiological processes such as cell proliferation, inflammation, differentiation, angiogenesis, transformation and apoptosis, pre-mRNA splicing, metabolic syndromes, fibrosis, and host defense. The role of Gal-3 has also been implicated in liver diseases. Gal-3 is activated upon a hepatotoxic insult to the liver and its level has been shown to be upregulated in fatty liver diseases, inflammation, nonalcoholic steatohepatitis, fibrosis, cholangitis, cirrhosis, and hepatocellular carcinoma (HCC). Gal-3 directly interacts with the NOD-like receptor family, pyrin domain containing 3, and activates the inflammasome in macrophages of the liver. In the chronically injured liver, Gal-3 secreted by injured hepatocytes and immune cells, activates hepatic stellate cells (HSCs) in a paracrine fashion to acquire a myofibroblast like collagen-producing phenotype. Activated HSCs in the fibrotic liver secrete Gal-3 which acts via autocrine signaling to exacerbate extracellular matrix synthesis and fibrogenesis. In the stromal microenvironment, Gal-3 activates cancer cell proliferation, migration, invasiveness, and metastasis. Clinically, increased serum levels and Gal-3 expression were observed in the liver tissue of nonalcoholic steatohepatitis, fibrotic/cirrhotic, and HCC patients. The pathological role of Gal-3 has been experimentally and clinically reported in the progression of chronic liver disease. Therefore, this review discusses the pathological role of Gal-3 in the progression of chronic liver diseases.
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Affiliation(s)
- Devaraj Ezhilarasan
- Department of Pharmacology, Molecular Medicine and Toxicology Lab, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Chennai, Tamil Nadu, India
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25
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Reeves AE, Huang ML. Proximity labeling technologies to illuminate glycan-protein interactions. Curr Opin Chem Biol 2023; 72:102233. [PMID: 36493526 PMCID: PMC9870929 DOI: 10.1016/j.cbpa.2022.102233] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2022] [Revised: 11/01/2022] [Accepted: 11/03/2022] [Indexed: 12/12/2022]
Abstract
Glycosylation is a ubiquitous post-translational modification read by glycan-binding proteins (GBP) to encode important functions, but a robust understanding of these interactions and their consequences can be challenging to uncover. Glycan-GBP interactions are transient and weak, making them difficult to capture, and glycosylation is dynamic and heterogenous, necessitating study in native cellular environments to identify endogenous ligands. Proximity labeling, an experimental innovation that labels biomolecules close to a protein of interest, has recently emerged as a powerful strategy to overcome these limitations, allowing interactors to be tagged in cells for subsequent enrichment and identification by mass spectrometry-based proteomics. We will describe this nascent technique and discuss its applications in the last five years with different GBP classes, including Siglecs, galectins, and non-human lectins.
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Affiliation(s)
- Abigail E Reeves
- Skaggs Graduate School of Chemical and Biological Sciences, Scripps Research, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA; Department of Molecular Medicine, Scripps Research, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA
| | - Mia L Huang
- Skaggs Graduate School of Chemical and Biological Sciences, Scripps Research, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA; Department of Molecular Medicine, Scripps Research, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA.
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26
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Vilen Z, Reeves AE, Huang ML. (Glycan Binding) Activity‐Based Protein Profiling in Cells Enabled by Mass Spectrometry‐Based Proteomics. Isr J Chem 2023; 63. [PMID: 37131487 PMCID: PMC10150848 DOI: 10.1002/ijch.202200097] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
The presence of glycan modifications at the cell surface and other locales positions them as key regulators of cell recognition and function. However, due to the complexity of glycosylation, the annotation of which proteins bear glycan modifications, which glycan patterns are present, and which proteins are capable of binding glycans is incomplete. Inspired by activity-based protein profiling to enrich for proteins in cells based on select characteristics, these endeavors have been greatly advanced by the development of appropriate glycan-binding and glycan-based probes. Here, we provide context for these three problems and describe how the capability of molecules to interact with glycans has enabled the assignment of proteins with specific glycan modifications or of proteins that bind glycans. Furthermore, we discuss how the integration of these probes with high resolution mass spectrometry-based technologies has greatly advanced glycoscience.
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Affiliation(s)
- Zak Vilen
- Skaggs Graduate School of Chemical and Biological Sciences Scripps Research 10550 N. Torrey Pines Rd. La Jolla CA 92037 USA
- Department of Molecular Medicine Scripps Research 10550 N. Torrey Pines Rd. La Jolla CA 92037, USA
| | - Abigail E. Reeves
- Skaggs Graduate School of Chemical and Biological Sciences Scripps Research 10550 N. Torrey Pines Rd. La Jolla CA 92037 USA
- Department of Molecular Medicine Scripps Research 10550 N. Torrey Pines Rd. La Jolla CA 92037, USA
| | - Mia L. Huang
- Skaggs Graduate School of Chemical and Biological Sciences Scripps Research 10550 N. Torrey Pines Rd. La Jolla CA 92037 USA
- Department of Molecular Medicine Scripps Research 10550 N. Torrey Pines Rd. La Jolla CA 92037, USA
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27
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Laderach DJ, Compagno D. Inhibition of galectins in cancer: Biological challenges for their clinical application. Front Immunol 2023; 13:1104625. [PMID: 36703969 PMCID: PMC9872792 DOI: 10.3389/fimmu.2022.1104625] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2022] [Accepted: 12/16/2022] [Indexed: 01/11/2023] Open
Abstract
Galectins play relevant roles in tumor development, progression and metastasis. Accordingly, galectins are certainly enticing targets for medical intervention in cancer. To date, however, clinical trials based on galectin inhibitors reported inconclusive results. This review summarizes the galectin inhibitors currently being evaluated and discusses some of the biological challenges that need to be addressed to improve these strategies for the benefit of cancer patients.
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Affiliation(s)
- Diego José Laderach
- Molecular and Functional Glyco-Oncology Laboratory, Instituto de Química Biológica de la Facutad de Ciencias Exactas y Naturales (IQUIBICEN-CONICET), Buenos Aires, Argentina,Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina,Departamento de Ciencias Básicas, Universidad Nacional de Luján, Luján, Argentina,*Correspondence: Diego José Laderach,
| | - Daniel Compagno
- Molecular and Functional Glyco-Oncology Laboratory, Instituto de Química Biológica de la Facutad de Ciencias Exactas y Naturales (IQUIBICEN-CONICET), Buenos Aires, Argentina,Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
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28
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Meyer CF, Seath CP, Knutson SD, Lu W, Rabinowitz JD, MacMillan DWC. Photoproximity Labeling of Sialylated Glycoproteins (GlycoMap) Reveals Sialylation-Dependent Regulation of Ion Transport. J Am Chem Soc 2022; 144:23633-23641. [PMID: 36525649 PMCID: PMC10782853 DOI: 10.1021/jacs.2c11094] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Sialylation, the addition of sialic acid to glycans, is a crucial post-translational modification of proteins, contributing to neurodevelopment, oncogenesis, and immune response. In cancer, sialylation is dramatically upregulated. Yet, the functional biochemical consequences of sialylation remain mysterious. Here, we establish a μMap proximity labeling platform that utilizes metabolically inserted azidosialic acid to introduce iridium-based photocatalysts on sialylated cell-surface glycoproteins as a means to profile local microenvironments across the sialylated proteome. In comparative experiments between primary cervical cells and a cancerous cell line (HeLa), we identify key differences in both the global sialome and proximal proteins, including solute carrier proteins that regulate metabolite and ion transport. In particular, we show that cell-surface interactions between receptors trafficking ethanolamine and zinc are sialylation-dependent and impact intracellular metabolite levels. These results establish a μMap method for interrogating proteoglycan function and support a role for sialylated glycoproteins in regulating cell-surface transporters.
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Affiliation(s)
- Claudio F. Meyer
- Merck Center for Catalysis at Princeton University, Princeton, New Jersey 08544, United States
- Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States
| | - Ciaran P. Seath
- Merck Center for Catalysis at Princeton University, Princeton, New Jersey 08544, United States
- Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States
| | - Steve D. Knutson
- Merck Center for Catalysis at Princeton University, Princeton, New Jersey 08544, United States
- Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States
| | - Wenyun Lu
- Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544, United States
| | - Joshua D. Rabinowitz
- Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544, United States
- Ludwig Institute for Cancer Research, Princeton University, Princeton, New Jersey, 08544, United States
| | - David W. C. MacMillan
- Merck Center for Catalysis at Princeton University, Princeton, New Jersey 08544, United States
- Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States
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29
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Fei F, Zhang M, Tarighat SS, Joo EJ, Yang L, Heisterkamp N. Galectin-1 and Galectin-3 in B-Cell Precursor Acute Lymphoblastic Leukemia. Int J Mol Sci 2022; 23:ijms232214359. [PMID: 36430839 PMCID: PMC9694201 DOI: 10.3390/ijms232214359] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2022] [Revised: 11/09/2022] [Accepted: 11/16/2022] [Indexed: 11/22/2022] Open
Abstract
Acute lymphoblastic leukemias arising from the malignant transformation of B-cell precursors (BCP-ALLs) are protected against chemotherapy by both intrinsic factors as well as by interactions with bone marrow stromal cells. Galectin-1 and Galectin-3 are lectins with overlapping specificity for binding polyLacNAc glycans. Both are expressed by bone marrow stromal cells and by hematopoietic cells but show different patterns of expression, with Galectin-3 dynamically regulated by extrinsic factors such as chemotherapy. In a comparison of Galectin-1 x Galectin-3 double null mutant to wild-type murine BCP-ALL cells, we found reduced migration, inhibition of proliferation, and increased sensitivity to drug treatment in the double knockout cells. Plant-derived carbohydrates GM-CT-01 and GR-MD-02 were used to inhibit extracellular Galectin-1/-3 binding to BCP-ALL cells in co-culture with stromal cells. Treatment with these compounds attenuated migration of the BCP-ALL cells to stromal cells and sensitized human BCP-ALL cells to vincristine and the targeted tyrosine kinase inhibitor nilotinib. Because N-glycan sialylation catalyzed by the enzyme ST6Gal1 can regulate Galectin cell-surface binding, we also compared the ability of BCP-ALL wild-type and ST6Gal1 knockdown cells to resist vincristine treatment when they were co-cultured with Galectin-1 or Galectin-3 knockout stromal cells. Consistent with previous results, stromal Galectin-3 was important for maintaining BCP-ALL fitness during chemotherapy exposure. In contrast, stromal Galectin-1 did not significantly contribute to drug resistance, and there was no clear effect of ST6Gal1-catalysed N-glycan sialylation. Taken together, our results indicate a complicated joint contribution of Galectin-1 and Galectin-3 to BCP-ALL survival, with different roles for endogenous and stromal produced Galectins. These data indicate it will be important to efficiently block both extracellular and intracellular Galectin-1 and Galectin-3 with the goal of reducing BCP-ALL persistence in the protective bone marrow niche during chemotherapy.
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Affiliation(s)
- Fei Fei
- Section of Molecular Carcinogenesis, Department of Pediatrics, Division of Hematology/Oncology and Bone Marrow Transplantation, The Saban Research Institute of Children’s Hospital, Los Angeles, CA 90027, USA
| | - Mingfeng Zhang
- Department of Systems Biology, Beckman Research Institute City of Hope, Monrovia, CA 91016, USA
| | - Somayeh S. Tarighat
- Section of Molecular Carcinogenesis, Department of Pediatrics, Division of Hematology/Oncology and Bone Marrow Transplantation, The Saban Research Institute of Children’s Hospital, Los Angeles, CA 90027, USA
| | - Eun Ji Joo
- Department of Systems Biology, Beckman Research Institute City of Hope, Monrovia, CA 91016, USA
| | - Lu Yang
- Department of Systems Biology, Beckman Research Institute City of Hope, Monrovia, CA 91016, USA
| | - Nora Heisterkamp
- Department of Systems Biology, Beckman Research Institute City of Hope, Monrovia, CA 91016, USA
- Correspondence: ; Tel.: +1-626-218-7503
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30
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Bhattacharya S, Zhang M, Hu W, Qi T, Heisterkamp N. Targeting disordered-structured domain interactions in Galectin-3 based on NMR and enhanced MD. Biophys J 2022; 121:4342-4357. [PMID: 36209362 PMCID: PMC9703043 DOI: 10.1016/j.bpj.2022.10.008] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2021] [Revised: 05/18/2022] [Accepted: 10/06/2022] [Indexed: 12/14/2022] Open
Abstract
Intrinsically disordered regions (IDRs) are common and important functional domains in many proteins. However, IDRs are difficult to target for drug development due to the lack of defined structures that would facilitate the identification of possible drug-binding pockets. Galectin-3 is a carbohydrate-binding protein of which overexpression has been implicated in a wide variety of disorders, including cancer and inflammation. Apart from its carbohydrate-recognition/binding domain (CRD), Galectin-3 also contains a functionally important disordered N-terminal domain (NTD) that contacts the C-terminal domain (CTD) and could be a target for drug development. To overcome challenges involved in inhibitor design due to lack of structure and the highly dynamic nature of the NTD, we used a protocol combining nuclear magnetic resonance data from recombinant Galectin-3 with accelerated molecular dynamics (MD) simulations. This approach identified a pocket in the CTD with which the NTD makes frequent contact. In accordance with this model, mutation of residues L131 and L203 in this pocket caused loss of Galectin-3 agglutination ability, signifying the functional relevance of the cavity. In silico screening was used to design candidate inhibitory peptides targeting the newly discovered cavity, and experimental testing of only three of these yielded one peptide that inhibits the agglutination promoted by wild-type Galectin-3. NMR experiments further confirmed that this peptide indeed binds to a cavity in the CTD, not within the actual CRD. Our results show that it is possible to apply a combination of MD simulations and NMR experiments to precisely predict the binding interface of a disordered domain with a structured domain, and furthermore use this predicted interface for designing inhibitors. This procedure can potentially be extended to many other targets in which similar IDR interactions play a vital functional role.
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Affiliation(s)
- Supriyo Bhattacharya
- Integrative Genomics Core, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA
| | - Mingfeng Zhang
- Department of Systems Biology, Beckman Research Institute City of Hope, Monrovia, California
| | - Weidong Hu
- Department of Molecular Imaging and Therapy, Beckman Research Institute of City of Hope, Duarte, California
| | - Tong Qi
- Department of Systems Biology, Beckman Research Institute City of Hope, Monrovia, California
| | - Nora Heisterkamp
- Department of Systems Biology, Beckman Research Institute City of Hope, Monrovia, California.
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31
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Switching of Photocatalytic Tyrosine/Histidine Labeling and Application to Photocatalytic Proximity Labeling. Int J Mol Sci 2022; 23:ijms231911622. [PMID: 36232972 PMCID: PMC9569449 DOI: 10.3390/ijms231911622] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2022] [Revised: 09/27/2022] [Accepted: 09/29/2022] [Indexed: 11/07/2022] Open
Abstract
Weak and transient protein interactions are involved in dynamic biological responses and are an important research subject; however, methods to elucidate such interactions are lacking. Proximity labeling is a promising technique for labeling transient ligand–binding proteins and protein–protein interaction partners of analytes via an irreversible covalent bond. Expanding chemical tools for proximity labeling is required to analyze the interactome. We developed several photocatalytic proximity-labeling reactions mediated by two different mechanisms. We found that numerous dye molecules can function as catalysts for protein labeling. We also identified catalysts that selectively modify tyrosine and histidine residues and evaluated their mechanisms. Model experiments using HaloTag were performed to demonstrate photocatalytic proximity labeling. We found that both ATTO465, which catalyzes labeling by a single electron transfer, and BODIPY, which catalyzes labeling by singlet oxygen, catalyze proximity labeling in cells.
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32
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Guo Y, Jia W, Yang J, Zhan X. Cancer glycomics offers potential biomarkers and therapeutic targets in the framework of 3P medicine. Front Endocrinol (Lausanne) 2022; 13:970489. [PMID: 36072925 PMCID: PMC9441633 DOI: 10.3389/fendo.2022.970489] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/16/2022] [Accepted: 08/02/2022] [Indexed: 11/30/2022] Open
Abstract
Glycosylation is one of the most important post-translational modifications (PTMs) in a protein, and is the most abundant and diverse biopolymer in nature. Glycans are involved in multiple biological processes of cancer initiation and progression, including cell-cell interactions, cell-extracellular matrix interactions, tumor invasion and metastasis, tumor angiogenesis, and immune regulation. As an important biomarker, tumor-associated glycosylation changes have been extensively studied. This article reviews recent advances in glycosylation-based biomarker research, which is useful for cancer diagnosis and prognostic assessment. Truncated O-glycans, sialylation, fucosylation, and complex branched structures have been found to be the most common structural patterns in malignant tumors. In recent years, immunochemical methods, lectin recognition-based methods, mass spectrometry (MS)-related methods, and fluorescence imaging-based in situ methods have greatly promoted the discovery and application potentials of glycomic and glycoprotein biomarkers in various cancers. In particular, MS-based proteomics has significantly facilitated the comprehensive research of extracellular glycoproteins, increasing our understanding of their critical roles in regulating cellular activities. Predictive, preventive and personalized medicine (PPPM; 3P medicine) is an effective approach of early prediction, prevention and personalized treatment for different patients, and it is known as the new direction of medical development in the 21st century and represents the ultimate goal and highest stage of medical development. Glycosylation has been revealed to have new diagnostic, prognostic, and even therapeutic potentials. The purpose of glycosylation analysis and utilization of biology is to make a fundamental change in health care and medical practice, so as to lead medical research and practice into a new era of 3P medicine.
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Affiliation(s)
- Yuna Guo
- Shandong Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University, Jinan, China
- Medical Science and Technology Innovation Center, Shandong First Medical University, Jinan, China
| | - Wenshuang Jia
- Shandong Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University, Jinan, China
- Medical Science and Technology Innovation Center, Shandong First Medical University, Jinan, China
| | - Jingru Yang
- Shandong Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University, Jinan, China
- Medical Science and Technology Innovation Center, Shandong First Medical University, Jinan, China
| | - Xianquan Zhan
- Shandong Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University, Jinan, China
- Medical Science and Technology Innovation Center, Shandong First Medical University, Jinan, China
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33
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Wisnovsky S, Bertozzi CR. Reading the glyco-code: New approaches to studying protein-carbohydrate interactions. Curr Opin Struct Biol 2022; 75:102395. [PMID: 35653954 PMCID: PMC9811956 DOI: 10.1016/j.sbi.2022.102395] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2022] [Revised: 04/06/2022] [Accepted: 04/16/2022] [Indexed: 01/07/2023]
Abstract
The surface of all living cells is decorated with carbohydrate molecules. Hundreds of functional proteins bind to these glycosylated ligands; such binding events subsequently modulate many aspects of protein and cell function. Identifying ligands for glycan-binding proteins (GBPs) is a defining challenge of glycoscience research. Here, we review recent advances that are allowing protein-carbohydrate interactions to be dissected with an unprecedented level of precision. We specifically highlight how cell-based glycan arrays and glyco-genomic profiling are being used to define the structural determinants of glycan-protein interactions in living cells. Going forward, these methods create exciting new opportunities for the study of glycans in physiology and disease.
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Affiliation(s)
- Simon Wisnovsky
- Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
| | - Carolyn R Bertozzi
- Department of Chemistry, Stanford University, Stanford, CA, 94305, USA; Howard Hughes Medical Institute, Stanford, CA, 94305, USA.
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34
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Griffin ME, Hsieh-Wilson LC. Tools for mammalian glycoscience research. Cell 2022; 185:2657-2677. [PMID: 35809571 PMCID: PMC9339253 DOI: 10.1016/j.cell.2022.06.016] [Citation(s) in RCA: 40] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2022] [Revised: 06/08/2022] [Accepted: 06/08/2022] [Indexed: 10/17/2022]
Abstract
Cellular carbohydrates or glycans are critical mediators of biological function. Their remarkably diverse structures and varied activities present exciting opportunities for understanding many areas of biology. In this primer, we discuss key methods and recent breakthrough technologies for identifying, monitoring, and manipulating glycans in mammalian systems.
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Affiliation(s)
- Matthew E Griffin
- Department of Chemistry, University of California, Irvine, Irvine, CA 92697, USA; Department of Microbiology and Immunology, Scripps Research, La Jolla, CA 92037, USA
| | - Linda C Hsieh-Wilson
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
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35
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Huang Y, Zhai G, Li Y, Han Y, Chen C, Lu C, Zhang K. Deciphering the Interactome of Histone Marks in Living Cells via Genetic Code Expansion Combined with Proximity Labeling. Anal Chem 2022; 94:10705-10714. [PMID: 35862615 DOI: 10.1021/acs.analchem.2c01042] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Deciphering the endogenous interactors of histone post-translational modifications (hPTMs, also called histone marks) is essential to understand the mechanisms of epigenetic regulation. However, most of the analytical methods to determine hPTM interactomes are in vitro settings, lacking interrogating native chromatin. Although lysine crotonylation (Kcr) has recently been considered an important hPTM for the regulation of gene transcription, the interactors of Kcr still remain to be explored. Herein, we present a general approach relying upon a genetic code expansion system, APEX2 (engineered peroxidase)-mediated proximity labeling, and quantitative proteomics to profile interactomes of the selected hPTMs in living cells. We genetically fused APEX2 to the recombinant histone H3 with a crotonyl lysine inserted site specifically to generate APEX2-H3K9cr that incorporated into native chromatin. Upon activation, APEX2 triggered in vivo biotin labeling of H3K9cr interactors that can then be enriched with streptavidin beads and identified by mass spectrometry. Proteomic analysis further revealed the endogenous interactomes of H3K9cr and confirmed the reliability of the method. Moreover, DPF2 was identified as a candidate interactor, and the binding interaction of DPF2 to H3K9c was further characterized and verified. This study provides a novel strategy for the identification of hPTM interactomes in living cells, and we envision that this is key to elucidating epigenetic regulatory pathways.
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Affiliation(s)
- Yepei Huang
- The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Key Laboratory of Immune Micro-environment and Disease (Ministry of Education), Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Department of Gastroenterology and Hepatology, Tianjin Medical University General Hospital, Tianjin Medical University, Tianjin 300070, China
| | - Guijin Zhai
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China
| | - Yanan Li
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China
| | - Yue Han
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China
| | - Chen Chen
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China
| | - Congcong Lu
- College of Life Sciences, Nankai University, Tianjin 300070, China
| | - Kai Zhang
- The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Key Laboratory of Immune Micro-environment and Disease (Ministry of Education), Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Department of Gastroenterology and Hepatology, Tianjin Medical University General Hospital, Tianjin Medical University, Tianjin 300070, China
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36
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Liu Y, Nelson ZM, Reda A, Fehl C. Spatiotemporal Proximity Labeling Tools to Track GlcNAc Sugar-Modified Functional Protein Hubs during Cellular Signaling. ACS Chem Biol 2022; 17:2153-2164. [PMID: 35819414 DOI: 10.1021/acschembio.2c00282] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
A fundamental mechanism that all eukaryotic cells use to adapt to their environment is dynamic protein modification with monosaccharide sugars. In humans, O-linked N-acetylglucosamine (O-GlcNAc) is rapidly added to and removed from diverse protein sites as a response to fluctuating nutrient levels, stressors, and signaling cues. Two aspects remain challenging for tracking functional O-GlcNAc events with chemical strategies: spatial control over subcellular locations and time control during labeling. The objective of this study was to create intracellular proximity labeling tools to identify functional changes in O-GlcNAc patterns with spatiotemporal control. We developed a labeling strategy based on the TurboID proximity labeling system for rapid protein biotin conjugation directed to O-GlcNAc protein modifications inside cells, a set of tools called "GlycoID." Localized variants to the nucleus and cytosol, nuc-GlycoID and cyt-GlycoID, labeled O-GlcNAc proteins and their interactomes in subcellular space. Labeling during insulin and serum stimulation revealed functional changes in O-GlcNAc proteins as soon as 30 min following signal initiation. We demonstrated using proteomic analysis that the GlycoID strategy captured O-GlcNAcylated "activity hubs" consisting of O-GlcNAc proteins and their associated protein-protein interactions. The ability to follow changes in O-GlcNAc hubs during physiological events such as insulin signaling allows these tools to determine the mechanisms of glycobiological cell regulation. Our functional O-GlcNAc data sets in human cells will be a valuable resource for O-GlcNAc-driven mechanisms.
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Affiliation(s)
- Yimin Liu
- Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202, United States
| | - Zachary M Nelson
- Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202, United States
| | - Ali Reda
- Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202, United States
| | - Charlie Fehl
- Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202, United States
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37
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Critcher M, Huang ML. Excavating proteoglycan structure-function relationships: Modern approaches to capture the interactions of ancient biomolecules. Am J Physiol Cell Physiol 2022; 323:C415-C422. [PMID: 35759439 PMCID: PMC9359657 DOI: 10.1152/ajpcell.00222.2022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Proteoglycans are now well regarded as key facilitators of cell biology. While a majority of their interactions and functions are attributed to the decorating glycosaminoglycan chains, there is a growing appreciation for the roles of the proteoglycan core protein and for considering proteoglycans as replete protein-glycan conjugates. This appreciation, seeded by early work in proteoglycan biology, is now being advanced and exalted by modern approaches in chemical glycobiology. In this review, we discuss up-and-coming methods to unearth the fine-scale architecture of proteoglycans that modulate their functions and interactions. Crucial to these efforts is the production of chemically defined materials, including semi-synthetic proteoglycans and the in situ capture of interacting proteins. Together, the integration of chemical biology approaches promises to expedite the dissection of the structural heterogeneity of proteoglycans and deliver refined insight into their functions.
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Affiliation(s)
- Meg Critcher
- Skaggs Graduate School of Chemical and Biological Sciences, Scripps Research, La Jolla, CA.,Department of Molecular Medicine, Scripps Research, La Jolla, CA
| | - Mia L Huang
- Skaggs Graduate School of Chemical and Biological Sciences, Scripps Research, La Jolla, CA.,Department of Molecular Medicine, Scripps Research, La Jolla, CA.,Department of Chemistry, Scripps Research, La Jolla, CA
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38
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O'Leary TR, Critcher M, Stephenson TN, Yang X, Hassan AA, Bartfield NM, Hawkins R, Huang ML. Chemical editing of proteoglycan architecture. Nat Chem Biol 2022; 18:634-642. [PMID: 35551261 PMCID: PMC9205196 DOI: 10.1038/s41589-022-01023-5] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2021] [Accepted: 03/29/2022] [Indexed: 12/21/2022]
Abstract
Proteoglycans are heterogeneous macromolecular glycoconjugates that orchestrate many important cellular processes. While much attention has focused on the poly-sulfated glycosaminoglycan chains that decorate proteoglycans, other important elements of their architecture, such as core proteins and membrane localization, have garnered less emphasis. Hence, comprehensive structure-function relationships that consider the replete proteoglycan architecture as glycoconjugates are limited. Here we present an extensive approach to study proteoglycan structure and biology by fabricating defined semisynthetic modular proteoglycans that can be tailored for cell surface display. The expression of proteoglycan core proteins with unnatural amino acids permits bioorthogonal click chemistry with functionalized glycosaminoglycans for methodical dissection of the parameters required for optimal binding and function of various proteoglycan-binding proteins. We demonstrate that these sophisticated materials can recapitulate the functions of native proteoglycan ectodomains in mouse embryonic stem cell differentiation and cancer cell spreading while permitting the analysis of the contributing architectural elements toward function.
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Affiliation(s)
- Timothy R O'Leary
- Department of Molecular Medicine, Scripps Research, Jupiter, FL, USA
| | - Meg Critcher
- Department of Molecular Medicine, Scripps Research, Jupiter, FL, USA
- Skaggs Graduate School of Chemical and Biological Sciences, Scripps Research, La Jolla, CA, USA
| | | | - Xueyi Yang
- Department of Molecular Medicine, Scripps Research, Jupiter, FL, USA
- Skaggs Graduate School of Chemical and Biological Sciences, Scripps Research, La Jolla, CA, USA
| | - Abdullah A Hassan
- Department of Molecular Medicine, Scripps Research, Jupiter, FL, USA
| | - Noah M Bartfield
- Department of Molecular Medicine, Scripps Research, Jupiter, FL, USA
| | - Richard Hawkins
- Department of Molecular Medicine, Scripps Research, Jupiter, FL, USA
| | - Mia L Huang
- Department of Molecular Medicine, Scripps Research, Jupiter, FL, USA.
- Skaggs Graduate School of Chemical and Biological Sciences, Scripps Research, La Jolla, CA, USA.
- Department of Molecular Medicine, Scripps Research, La Jolla, CA, USA.
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39
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Critcher M, Hassan AA, Huang ML. Seeing the forest through the trees: characterizing the glycoproteome. Trends Biochem Sci 2022; 47:492-505. [DOI: 10.1016/j.tibs.2022.02.007] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2021] [Revised: 02/10/2022] [Accepted: 02/21/2022] [Indexed: 12/14/2022]
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40
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Jiang HS, Zhuang SC, Lam CH, Chang LY, Angata T. Recent Progress in the Methodologies to Identify Physiological Ligands of Siglecs. Front Immunol 2021; 12:813082. [PMID: 34956244 PMCID: PMC8702521 DOI: 10.3389/fimmu.2021.813082] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2021] [Accepted: 11/23/2021] [Indexed: 11/28/2022] Open
Abstract
Siglecs, a family of receptor-like lectins, recognize glycoproteins and/or glycolipids containing sialic acid in the extracellular space and transduce intracellular signaling. Recently, researchers uncovered significant contributions of Siglecs in cancer immunity, renewing interest in this family of proteins. Previous extensive studies have defined how Siglecs recognize glycan epitopes (glycotopes). Nevertheless, the biological role of these glycotopes has not been fully evaluated. Recent studies using live cells have begun unraveling the constituents of Siglec ligands. These studies demonstrated that glycoprotein scaffolds (counter-receptors) displaying glycotopes are sometimes just as important as the glycotope itself. These new insights may guide future efforts to develop therapeutic agents to target the Siglec – ligand axis.
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Affiliation(s)
- Huei-Syuan Jiang
- Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.,Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan.,Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
| | - Shao-Chien Zhuang
- Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.,Genomics Research Center, Academia Sinica, Taipei, Taiwan
| | - Chak Hin Lam
- Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
| | - Lan-Yi Chang
- Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
| | - Takashi Angata
- Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.,Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan.,Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
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41
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Tarighat SS, Fei F, Joo EJ, Abdel-Azim H, Yang L, Geng H, Bum-Erdene K, Grice ID, von Itzstein M, Blanchard H, Heisterkamp N. Overcoming Microenvironment-Mediated Chemoprotection through Stromal Galectin-3 Inhibition in Acute Lymphoblastic Leukemia. Int J Mol Sci 2021; 22:12167. [PMID: 34830047 PMCID: PMC8624256 DOI: 10.3390/ijms222212167] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2021] [Revised: 11/06/2021] [Accepted: 11/08/2021] [Indexed: 11/17/2022] Open
Abstract
Environmentally-mediated drug resistance in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) significantly contributes to relapse. Stromal cells in the bone marrow environment protect leukemia cells by secretion of chemokines as cues for BCP-ALL migration towards, and adhesion to, stroma. Stromal cells and BCP-ALL cells communicate through stromal galectin-3. Here, we investigated the significance of stromal galectin-3 to BCP-ALL cells. We used CRISPR/Cas9 genome editing to ablate galectin-3 in stromal cells and found that galectin-3 is dispensable for steady-state BCP-ALL proliferation and viability. However, efficient leukemia migration and adhesion to stromal cells are significantly dependent on stromal galectin-3. Importantly, the loss of stromal galectin-3 production sensitized BCP-ALL cells to conventional chemotherapy. We therefore tested novel carbohydrate-based small molecule compounds (Cpd14 and Cpd17) with high specificity for galectin-3. Consistent with results obtained using galectin-3-knockout stromal cells, treatment of stromal-BCP-ALL co-cultures inhibited BCP-ALL migration and adhesion. Moreover, these compounds induced anti-leukemic responses in BCP-ALL cells, including a dose-dependent reduction of viability and proliferation, the induction of apoptosis and, importantly, the inhibition of drug resistance. Collectively, these findings indicate galectin-3 regulates BCP-ALL cell responses to chemotherapy through the interactions between leukemia cells and the stroma, and show that a combination of galectin-3 inhibition with conventional drugs can sensitize the leukemia cells to chemotherapy.
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Affiliation(s)
- Somayeh S. Tarighat
- Division of Hematology/Oncology and Bone Marrow Transplant, The Saban Research Institute of Children’s Hospital Los Angeles, Los Angeles, CA 90027, USA; (S.S.T.); (F.F.); (E.J.J.); (H.A.-A.)
| | - Fei Fei
- Division of Hematology/Oncology and Bone Marrow Transplant, The Saban Research Institute of Children’s Hospital Los Angeles, Los Angeles, CA 90027, USA; (S.S.T.); (F.F.); (E.J.J.); (H.A.-A.)
| | - Eun Ji Joo
- Division of Hematology/Oncology and Bone Marrow Transplant, The Saban Research Institute of Children’s Hospital Los Angeles, Los Angeles, CA 90027, USA; (S.S.T.); (F.F.); (E.J.J.); (H.A.-A.)
- Department of Systems Biology, Beckman Research Institute, City of Hope, Monrovia, CA 91016, USA;
| | - Hisham Abdel-Azim
- Division of Hematology/Oncology and Bone Marrow Transplant, The Saban Research Institute of Children’s Hospital Los Angeles, Los Angeles, CA 90027, USA; (S.S.T.); (F.F.); (E.J.J.); (H.A.-A.)
| | - Lu Yang
- Department of Systems Biology, Beckman Research Institute, City of Hope, Monrovia, CA 91016, USA;
| | - Huimin Geng
- Department of Laboratory Medicine, University of California, San Francisco, CA 94143, USA;
| | - Khuchtumur Bum-Erdene
- Institute for Glycomics, Griffith University, Gold Coast, Southport, QLD 4222, Australia; (K.B.-E.); (I.D.G.); (M.v.I.); (H.B.)
| | - I. Darren Grice
- Institute for Glycomics, Griffith University, Gold Coast, Southport, QLD 4222, Australia; (K.B.-E.); (I.D.G.); (M.v.I.); (H.B.)
- School of Medical Science, Griffith University, Gold Coast, Southport, QLD 4222, Australia
| | - Mark von Itzstein
- Institute for Glycomics, Griffith University, Gold Coast, Southport, QLD 4222, Australia; (K.B.-E.); (I.D.G.); (M.v.I.); (H.B.)
| | - Helen Blanchard
- Institute for Glycomics, Griffith University, Gold Coast, Southport, QLD 4222, Australia; (K.B.-E.); (I.D.G.); (M.v.I.); (H.B.)
- School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, NSW 2522, Australia
- Illawarra Health & Medical Research Institute, Wollongong, NSW 2522, Australia
| | - Nora Heisterkamp
- Division of Hematology/Oncology and Bone Marrow Transplant, The Saban Research Institute of Children’s Hospital Los Angeles, Los Angeles, CA 90027, USA; (S.S.T.); (F.F.); (E.J.J.); (H.A.-A.)
- Department of Systems Biology, Beckman Research Institute, City of Hope, Monrovia, CA 91016, USA;
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42
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Vilen Z, Joeh E, Critcher M, Parker CG, Huang ML. Proximity Tagging Identifies the Glycan-Mediated Glycoprotein Interactors of Galectin-1 in Muscle Stem Cells. ACS Chem Biol 2021; 16:1994-2003. [PMID: 34181849 DOI: 10.1021/acschembio.1c00313] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Myogenic differentiation, the irreversible developmental process where precursor myoblast muscle stem cells become contractile myotubes, is heavily regulated by glycosylation and glycan-protein interactions at the cell surface and the extracellular matrix. The glycan-binding protein galectin-1 has been found to be a potent activator of myogenic differentiation. While it is being explored as a potential therapeutic for muscle repair, a precise understanding of its glycoprotein interactors is lacking. These gaps are due in part to the difficulties of capturing glycan-protein interactions in live cells. Here, we demonstrate the use of a proximity tagging strategy coupled with quantitative mass-spectrometry-based proteomics to capture, enrich, and identify the glycan-mediated glycoprotein interactors of galectin-1 in cultured live mouse myoblasts. Our interactome dataset can serve as a resource to aid the determination of mechanisms through which galectin-1 promotes myogenic differentiation. Moreover, it can also facilitate the determination of the physiological glycoprotein counter-receptors of galectin-1. Indeed, we identify several known and novel glycan-mediated ligands of galectin-1 as well as validate that galectin-1 binds the native CD44 glycoprotein in a glycan-mediated manner.
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Affiliation(s)
- Zak Vilen
- Department of Molecular Medicine, Scripps Research, 120 Scripps Way, Jupiter, Florida 33458-5284, United States
| | - Eugene Joeh
- Department of Molecular Medicine, Scripps Research, 120 Scripps Way, Jupiter, Florida 33458-5284, United States
| | - Meg Critcher
- Department of Molecular Medicine, Scripps Research, 120 Scripps Way, Jupiter, Florida 33458-5284, United States
| | - Christopher G. Parker
- Department of Chemistry, Scripps Research, 120 Scripps Way, Jupiter, Florida 33458-5284, United States
| | - Mia L. Huang
- Department of Molecular Medicine, Scripps Research, 120 Scripps Way, Jupiter, Florida 33458-5284, United States
- Department of Chemistry, Scripps Research, 120 Scripps Way, Jupiter, Florida 33458-5284, United States
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Lightfoot A, McGettrick HM, Iqbal AJ. Vascular Endothelial Galectins in Leukocyte Trafficking. Front Immunol 2021; 12:687711. [PMID: 34140956 PMCID: PMC8204101 DOI: 10.3389/fimmu.2021.687711] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2021] [Accepted: 05/12/2021] [Indexed: 12/16/2022] Open
Abstract
Leukocyte recruitment to the site of injury is a crucial event in the regulation of an inflammatory response. Tight regulation of interactions between the endothelium and circulating leukocytes is necessary to ensure a protective response to injury does not result in inflammatory disease. Rising interest in the broad immunoregulatory roles displayed by members of the glycan-binding galectin family suggests that these proteins could be an attractive target for therapeutic intervention, since their expression is significantly altered in disease. The focus of this review is to summarize current knowledge on the role of galectins in leukocyte trafficking during inflammation and the clinical approaches being taken to target these interactions for treatment of inflammatory disease.
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Affiliation(s)
- Abbey Lightfoot
- Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom
| | - Helen M McGettrick
- Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United Kingdom
| | - Asif J Iqbal
- Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom
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Abstract
Aberrant Ras signaling is linked to a wide spectrum of hyperproliferative diseases, and components of the signaling pathway, including Ras, have been the subject of intense and ongoing drug discovery efforts. The cellular activity of Ras is modulated by its association with the guanine nucleotide exchange factor Son of sevenless (Sos), and the high-resolution crystal structure of the Ras-Sos complex provides a basis for the rational design of orthosteric Ras ligands. We constructed a synthetic Sos protein mimic that engages the wild-type and oncogenic forms of nucleotide-bound Ras and modulates downstream kinase signaling. The Sos mimic was designed to capture the conformation of the Sos helix-loop-helix motif that makes critical contacts with Ras in its switch region. Chemoproteomic studies illustrate that the proteomimetic engages Ras and other cellular GTPases. The synthetic proteomimetic resists proteolytic degradation and enters cells through macropinocytosis. As such, it is selectively toxic to cancer cells with up-regulated macropinocytosis, including those that feature oncogenic Ras mutations.
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Zapatero-Belinchón FJ, Carriquí-Madroñal B, Gerold G. Proximity labeling approaches to study protein complexes during virus infection. Adv Virus Res 2021; 109:63-104. [PMID: 33934830 DOI: 10.1016/bs.aivir.2021.02.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Cellular compartmentalization of proteins and protein complex formation allow cells to tightly control biological processes. Therefore, understanding the subcellular localization and interactions of a specific protein is crucial to uncover its biological function. The advent of proximity labeling (PL) has reshaped cellular proteomics in infection biology. PL utilizes a genetically modified enzyme that generates a "labeling cloud" by covalently labeling proteins in close proximity to the enzyme. Fusion of a PL enzyme to a specific antibody or a "bait" protein of interest in combination with affinity enrichment mass spectrometry (AE-MS) enables the isolation and identification of the cellular proximity proteome, or proxisome. This powerful methodology has been paramount for the mapping of membrane or membraneless organelles as well as for the understanding of hard-to-purify protein complexes, such as those of transmembrane proteins. Unsurprisingly, more and more infection biology research groups have recognized the potential of PL for the identification of host-pathogen interactions. In this chapter, we introduce the enzymes commonly used for PL labeling as well as recent promising advancements and summarize the major achievements in organelle mapping and nucleic acid PL. Moreover, we comprehensively describe the research on host-pathogen interactions using PL, giving special attention to studies in the field of virology.
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Affiliation(s)
- Francisco José Zapatero-Belinchón
- Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany; Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a Joint Venture Between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany; Department of Clinical Microbiology, Virology, Umeå University, Umeå, Sweden; Wallenberg Centre for Molecular Medicine (WCMM), Umeå University, Umeå, Sweden.
| | - Belén Carriquí-Madroñal
- Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany; Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a Joint Venture Between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany
| | - Gisa Gerold
- Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany; Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a Joint Venture Between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany; Department of Clinical Microbiology, Virology, Umeå University, Umeå, Sweden; Wallenberg Centre for Molecular Medicine (WCMM), Umeå University, Umeå, Sweden.
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Joeh E, Reeves AE, Parker CG, Huang ML. Mapping Interactions between Glycans and Glycan-Binding Proteins by Live Cell Proximity Tagging. Curr Protoc 2021; 1:e104. [PMID: 33861898 PMCID: PMC8274366 DOI: 10.1002/cpz1.104] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Interactions between glycans and glycan-binding proteins (GBPs) consist of weak, noncovalent, and transient binding events, making them difficult to study in live cells void of a static, isolated system. Furthermore, the glycans are often presented as protein glycoconjugates, but there are limited efforts to identify these proteins. Proximity labeling permits covalent tagging of the glycoprotein interactors to query GBP in live cells. Coupled with high-resolution mass spectrometry, it facilitates determination of the proteins bearing the interacting glycans. In this method, fusion protein constructs of a GBP of interest with a peroxidase enzyme allows for in situ spatiotemporal radical-mediated tagging of interacting glycoproteins in living cells that can be enriched for identification. Using this method, the capture and study of glycan-GBP interactions no longer relies on weak, transient interactions, and results in robust capture and identification of the interactome of a GBP while preserving the native cellular environment. This protocol focuses on (1) expression and characterization of a recombinant fusion protein consisting of a peroxidase and the GBP galectin-3, (2) corresponding in situ labeling and visualization of interactors, (3) and proteomic workflow and analysis of captured proteins for robust identification using mass spectrometry. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Expression, purification, and characterization of recombinant fusion protein Alternate Protocol 1: Manual Ni-NTA purification of recombinant fusion protein Basic Protocol 2: In situ proximity labeling and evaluation by fluorescence microscopy Alternate Protocol 2: Western blot analysis of in situ proximity labeling Basic Protocol 3: Proximity labeling of cells for quantitative MS-based proteomics with tandem mass tags.
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Affiliation(s)
- Eugene Joeh
- Department of Molecular Medicine, Scripps Research Institute, Jupiter, Florida
| | - Abigail E Reeves
- Department of Molecular Medicine, Scripps Research Institute, Jupiter, Florida
| | | | - Mia L Huang
- Department of Molecular Medicine, Scripps Research Institute, Jupiter, Florida.,Department of Chemistry, Scripps Research Institute, Jupiter, Florida
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Porębska N, Poźniak M, Matynia A, Żukowska D, Zakrzewska M, Otlewski J, Opaliński Ł. Galectins as modulators of receptor tyrosine kinases signaling in health and disease. Cytokine Growth Factor Rev 2021; 60:89-106. [PMID: 33863623 DOI: 10.1016/j.cytogfr.2021.03.004] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2021] [Revised: 03/12/2021] [Accepted: 03/16/2021] [Indexed: 12/11/2022]
Abstract
Receptor tyrosine kinases (RTKs) constitute a large group of cell surface proteins that mediate communication of cells with extracellular environment. RTKs recognize external signals and transfer information to the cell interior, modulating key cellular activities, like metabolism, proliferation, motility, or death. To ensure balanced stream of signals the activity of RTKs is tightly regulated by numerous mechanisms, including receptor expression and degradation, ligand specificity and availability, engagement of co-receptors, cellular trafficking of the receptors or their post-translational modifications. One of the most widespread post-translational modifications of RTKs is glycosylation of their extracellular domains. The sugar chains attached to RTKs form a new layer of information, so called glyco-code that is read by galectins, carbohydrate binding proteins. Galectins are family of fifteen lectins implicated in immune response, inflammation, cell division, motility and death. The versatility of cellular activities attributed to galectins is a result of their high abundance and diversity of their cellular targets. A various sugar specificity of galectins and the differential ability of galectin family members to form oligomers affect the spatial distribution and the function of their cellular targets. Importantly, galectins and RTKs are tightly linked to the development, progression and metastasis of various cancers. A growing number of studies points on the close cooperation between RTKs and galectins in eliciting specific cellular responses. This review focuses on the identified complexes between galectins and RTK members and discusses their relevance for the cell physiology both in healthy tissues and in cancer.
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Affiliation(s)
- Natalia Porębska
- Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383, Wroclaw, Poland
| | - Marta Poźniak
- Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383, Wroclaw, Poland
| | - Aleksandra Matynia
- Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383, Wroclaw, Poland
| | - Dominika Żukowska
- Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383, Wroclaw, Poland
| | - Małgorzata Zakrzewska
- Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383, Wroclaw, Poland
| | - Jacek Otlewski
- Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383, Wroclaw, Poland
| | - Łukasz Opaliński
- Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383, Wroclaw, Poland.
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Glycoengineering: scratching the surface. Biochem J 2021; 478:703-719. [DOI: 10.1042/bcj20200612] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2020] [Revised: 12/22/2020] [Accepted: 01/19/2021] [Indexed: 12/11/2022]
Abstract
At the surface of many cells is a compendium of glycoconjugates that form an interface between the cell and its surroundings; the glycocalyx. The glycocalyx serves several functions that have captivated the interest of many groups. Given its privileged residence, this meshwork of sugar-rich biomolecules is poised to transmit signals across the cellular membrane, facilitating communication with the extracellular matrix and mediating important signalling cascades. As a product of the glycan biosynthetic machinery, the glycocalyx can serve as a partial mirror that reports on the cell's glycosylation status. The glycocalyx can also serve as an information-rich barrier, withholding the entry of pathogens into the underlying plasma membrane through glycan-rich molecular messages. In this review, we provide an overview of the different approaches devised to engineer glycans at the cell surface, highlighting considerations of each, as well as illuminating the grand challenges that face the next era of ‘glyco-engineers’. While we have learned much from these techniques, it is evident that much is left to be unearthed.
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