Identification of Siglec Ligands Using a Proximity Labeling Method

Chang, Lan-Yi; Chen, Yi–Ju; Fan, Chan-Yo; Tang, Chin-Ju; Chen, Yi-Hsiu; Low, Penk-Yeir; Ventura, Alessandro; Lin, Chun‐Cheng; Chen, Yu‐Ju; Angata, Takashi

doi:10.1021/acs.jproteome.7b00625

Cited by 61 publications

(52 citation statements)

References 57 publications

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“…2E). While other labeling strategies use horse radish peroxidase fusion proteins [35] that require oxidative conditions, our method uses the APEX2 enzyme for covalent tagging. APEX2 is active in both oxidative extracellular conditions and the reducing environment of the intracellular milieu [36], permitting the identification of 554 interactors for PX-Gal3 when it is intracellularly over-expressed ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling

Joeh

O’Leary

et al. 2020

Preprint

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Galectin-3 is a glycan-binding protein (GBP) that binds b-galactoside glycan structures to orchestrate a variety of important biological events, including the activation of hepatic stellate cells to cause hepatic fibrosis. While the requisite glycan epitopes needed to bind galectin-3 have long been elucidated, the cellular glycoproteins that bear these glycan signatures remain unknown. Given the importance of the threedimensional arrangement of glycans in dictating GBP interactions, strategies that allow the identification of GBP receptors in live cells, where the native glycan presentation and glycoprotein expression are preserved, possess significant advantages over static and artificial systems. Here, we describe the integration of a proximity labeling method and quantitative mass spectrometry to map the glycan and glycoprotein interactors for galectin-3 in live hepatic stellate cells. Understanding the identity of the glycoproteins and defining the structures of the glycans required for galectin-3 mediated hepatic stellate cell activation will empower efforts to design and develop selective therapeutics to mitigate hepatic fibrosis. SignificanceBecause of the weak interactions between individual glycan-binding proteins (GBP), such as galectin-3, and glycans, strategies that allow the direct interrogation of these interactions in living cells remain limited.Thus, the glycan and glycoprotein ligands that are physiologically relevant for galectin-3 binding are insufficiently described. Here, we used a proximity labeling approach that catalytically tags interactors for galectin-3 and identified its pertinent glycan and glycoprotein counter-receptors in live hepatic stellate cells.This study demonstrates that proximity labeling is a powerful tool for mapping GBP complexes in living cells, and when coupled with chemical inhibitors, it can discriminate between protein-protein and proteinglycan interactions. Graphical Abstract

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Section: Discussionmentioning

confidence: 99%

Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling

Joeh

O’Leary

et al. 2020

Preprint

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“…6), we hypothesized the following: 1) interaction between mSiglec-14 and a cis-ligand, likely a pattern-recognition receptor, triggers low-level production of cytokines/chemokines by Siglec-14/THP-1 in the absence of microbial stimulation, and 2) sSiglec-14, by interfering with the interaction between mSiglec-14 and the cis-ligand, cancels the enhanced production of cytokines/chemokines. To detect how sSiglec-14 suppresses production of pro-inflammatory cytokines/chemokines by Siglec-14/THP-1 cells, we sought to identify a protein that interacts with mSiglec-14 by a proximity labeling method (25). As shown in Fig.…”

Section: Ssiglec-14 Interferes With the Interaction Between Msiglec-1mentioning

confidence: 99%

“…Proximity biotin labeling and identification of proteins interacting with mSiglec-14 were performed by applying the method previously described (25). FLAG-Siglec-14/THP-1 cells (5 ϫ 10 6 cells) were incubated with 5 g of anti-FLAG M2-peroxidase antibody (A8592, Sigma) on ice for 30 min and washed twice with PBS.…”

Section: Proximity Biotin Labeling and Identification Of Siglec-14-inmentioning

confidence: 99%

“…Biotinylated proteins were purified with 200 l of Dynabeads MyOne Streptavidin C1 paramagnetic beads (ThermoFisher Scientific), eluted with 50 l of SDS-PAGE sample buffer, and subjected to brief SDS-PAGE. The gel area containing proteins was excised, in-gel trypsin-digested, and subjected to LC-coupled tandem MS for protein identification using a TLQ-Orbitrap Velos mass spectrometer (ThermoFisher Scientific), as described previously (25).…”

Section: Proximity Biotin Labeling and Identification Of Siglec-14-inmentioning

confidence: 99%

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Soluble Siglec-14 glycan-recognition protein is generated by alternative splicing and suppresses myeloid inflammatory responses

Huang

Low

Wang

et al. 2018

Journal of Biological Chemistry

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Human sialic acid-binding immunoglobulin-like lectin 14 (Siglec-14) is a glycan-recognition protein that is expressed on myeloid cells, recognizes bacterial pathogens, and elicits proinflammatory responses. Although Siglec-14 is a transmembrane protein, a soluble form of Siglec-14 is also present in human blood. However, the mechanism that generates soluble Siglec-14 and what role this protein form may play remain unknown. Here, investigating the generation and function of soluble Siglec-14, we found that soluble Siglec-14 is derived from an alternatively spliced mRNA that retains intron 5, containing a termination codon and thus preventing the translation of exon 6, which encodes Siglec-14's transmembrane domain. We also note that the translated segment in intron 5 encodes a unique C-terminal 7-amino acid extension, which allowed the specific antibody-mediated detection of this isoform in human blood. Moreover, soluble Siglec-14 dose-dependently suppressed pro-inflammatory responses of myeloid cells that expressed membrane-bound Siglec-14, likely by interfering with the interaction between membrane-bound Siglec-14 andToll-like receptor 2 on the cell surface. We also found that intron 5 contains a G-rich segment that assumes an RNA tertiary structure called a G-quadruplex, which may regulate the efficiency of intron 5 splicing. Taken together, we propose that soluble Siglec-14 suppresses pro-inflammatory responses triggered by membrane-bound Siglec-14. 3 The abbreviations used are: IL-6, interleukin-6; ADAM, a disintegrin and metalloproteinase domain-containing protein; COPD, chronic obstructive pulmonary disease; CXCL, CXC chemokine ligand; FBS, fetal bovine serum; hnRNP, heterogeneous nuclear

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“…The key role in modulating the regulatory activity of siglecs is associated with their ability to interact with ligands [34]. Since sialic acids are present in all cells, glycan ligands of siglecs are effective markers [35]. Discovering specific selective ligands for siglecs will make it possible to transport medicinal substances into the cell [36].…”

mentioning

confidence: 99%

Sialic Acid-Binding Lectins as Potential Pathophysiological Targets in Treatment of Chronic Bronchopulmonary Diseases (Review)

Kytikovа¹,

Gvozdenko²,

Аntonyuk³

et al. 2019

Sovrem Tehnol Med

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The article is devoted to the problem of finding the pathophysiological targets for optimizing the treatment of common and socially significant chronic respiratory diseases-bronchial asthma (BA) and chronic obstructive pulmonary disease (COPD). The balance between pro-inflammatory and anti-inflammatory mechanisms determines the nature of inflammation in these diseases. Among the regulators of inflammation there are siglecs-sialic acid-binding immunoglobulin-like lectins able to interact with terminal sialic acid present in all cells. Siglecs are involved in regulating cell proliferation, differentiation, apoptosis and implementing cell-cell interactions. The key role in modulating the regulatory activity of siglecs is in their ability to interact with ligands. Although research on the structure and biological functions of siglecs in the body has only recently started, the prospects for this research are promising, especially with respect to BA and COPD treatment. Siglecs are mainly expressed by immune and peripheral blood cells. Pathophysiological mechanisms of BA and COPD are implemented with participation of eosinophils, mast cells, neutrophils, and macrophages. The siglecs expressed on them play a particular role in the severity of tissue damage caused by the influence of these cells and therefore can be attractive targets for treatment of chronic inflammatory diseases of the respiratory organs. Siglec-8 and Siglec-10 molecules expressed on eosinophils have been actively studied in BA pathogenesis. However, given the importance of not only eosinophils, but also other cells in the disease pathogenesis, it seems challenging to investigate the role of Siglec-3, Siglec-5, Siglec-6, and Siglec-14 expressed on mast cells and basophils. In recent years, the role of Siglec-3, Siglec-9, and Siglec-5/14 has been studied in the pathogenesis of COPD. The use of antibodies against Siglec-15 described recently may be relevant in treatment of osteoporosis often associated with COPD. Based on scientific literature data, this article reviews the role of siglecs as possible regulators of inflammation in patients with chronic bronchopulmonary diseases.

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Identification of Siglec Ligands Using a Proximity Labeling Method

Cited by 61 publications

References 57 publications

Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling

Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling

Soluble Siglec-14 glycan-recognition protein is generated by alternative splicing and suppresses myeloid inflammatory responses

Sialic Acid-Binding Lectins as Potential Pathophysiological Targets in Treatment of Chronic Bronchopulmonary Diseases (Review)

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