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Glycoproteomic analysis of MGL binding protein on acute T cell leukemia cells

ABSTRACT: O-glycosylation is generally initiated by the transfer of a N-acetylgalactosamine to Ser/Thr residues of proteins, forming the Tn antigen. This truncated surface glycan is expressed at high levels by tumor cells and is associated with higher metastatic behaviour and poor prognosis of patients. The Tn antigen is recognised by the C-type macrophage galactose lectin (MGL), which induces the activation of immunosuppressive responses. Here, we investigated the MGL binding proteins in Jurkat cells. The optimization of pull-down assays and subsequent glycoproteomic analysis by mass spectrometry, allowed us to identify 17 cell surface proteins as novel MGL-ligands.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell, Cell Culture

DISEASE(S): Acute Leukemia

SUBMITTER: Yassene Mohammed

LAB HEAD: Paul Hensbergen

PROVIDER: PXD011307 | Pride | 2019-01-02

REPOSITORIES: Pride

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Glycoproteomic Analysis of MGL-Binding Proteins on Acute T-Cell Leukemia Cells.

Pirro Martina M Schoof Esmee E van Vliet Sandra J SJ Rombouts Yoann Y Stella Alexandre A de Ru Arnoud A Mohammed Yassene Y Wuhrer Manfred M van Veelen Peter A PA Hensbergen Paul J PJ

Journal of proteome research 20190109 3

C-type lectins are a diverse group of proteins involved in many human physiological and pathological processes. Most C-type lectins are glycan-binding proteins, some of which are pivotal for innate immune responses against pathogens. Other C-type lectins, such as the macrophage galactose-type lectin (MGL), have been shown to induce immunosuppressive responses upon the recognition of aberrant glycosylation on cancer cells. MGL is known to recognize terminal N-acetylgalactosamine (GalNAc), such as ...[more]

PMID: 30582698

Similar Datasets

Characterization of Macrophage Galactose-type Lectin (MGL) ligands in colorectal cancer cell lines

Project description:Background The Ca2+-dependent C-type lectin receptor Macrophage Galactose-type Lectin (MGL) is highly expressed by tolerogenic dendritic cells (DC) and macrophages. MGL exhibits a high binding specificity for terminal alpha- and beta-linked GalNAc residues found in Tn, sTn and LacdiNAc antigens. These glycan epitopes are often overexpressed in colorectal cancer (CRC), and, as such, MGL can be used to discriminate tumor from the corresponding healthy tissues. Moreover, the high expression of MGL ligands is associated with poor disease-free survival in stage III of CRC tumors. Nonetheless, the glycoproteins expressed by tumor cells that are recognized by MGL have hitherto remained elusive. Methods Using a panel of three CRC cell lines (HCT116, HT29 and LS174T), recapitulating CRC diversity, we performed FACS staining and pull-down assays using a recombinant soluble form of MGL (and a mutant MGL as control) combined with mass spectrometry-based (glyco)proteomics. Results HCT116 and HT29, but not LS174T, are high MGL-binding CRC cell lines. On these cells, the major cell surface binding proteins are receptors (e.g. MET, PTK7, SORL1, PTPRF) and integrins (ITGB1, ITGA3). From these proteins, several N- and/or O-glycopeptides were identified, of which some carried either a LacdiNAc or Tn epitope.

2020-01-09 | PXD016726 | Pride

Probing the ovarian cancer O-glycoproteome by the Macrophage Galactose like C-type Lectin (MGL): dissecting novel patterns of immune recognition

Project description:Glycosylation, the posttranslational linking of sugar molecules to proteins, is notoriously altered during tumor transformation. More specifically in carcinomas, GalNAc-type O-glycosylation, is characterized by biosynthetically immature truncated glycans present on the cancer cell surface which profoundly impacts anti-tumor immune recognition. The tumor associated glycan pattern may thus be regarded as a biomarker of immune modulation. In Epithelial Ovarian Cancer (EOC) there is a particular lack of specific biomarkers and molecular targets to aid early diagnose and develop novel therapeutic interventions, respectively. The aim of this study was therefore to interrogate the ovarian cancer O-glycoproteome and identify tumor associated glycoproteins relevant in tumor-Dendritic Cell (DC) interactions, mediated by the MGL C-type lectin, recognizing the tumor associated Tn O-glycan. Here we present a strategy, employing a recombinant human MGL protein, to probe the ovarian cancer O-glycoproteome. This was achieved by a MGL Lectin Weak Affinity Chromatography (LWAC) approach using glycoengineered ovarian cancer cells and ovarian cancer tissues as input material. Biochemical and bioinformatics analysis allowed us to identify the glycan structure/arrangement identified by MGL and furthermore to recognize potential MGL binders located at cell membrane, as expected, but also within the intracellular compartment and the matrisome, strongly suggesting that MGL in vivo could act as sensor of cellular distress/damage and modulator of DC motility. The tumor glycoproteins binders for MGL may become relevant as potential “onco-immune” biomarkers for EOC progression and prognosis. Furthermore, these results contribute to the design of glyco-immunogens for DC-based cancer immunotherapy.

2020-10-19 | PXD017510 | Pride

N-glycoproteins have a major role in MGL binding to colorectal cancer cell lines

Project description:Colorectal cancer (CRC) is the second leading cause of cancer death worldwide due in part to a high proportion of patients diagnosed at advanced stages of the disease. For this reason, many efforts have been made towards new approaches for early detection and prognosis. Cancer-associated aberrant glycosylation, especially the Tn and STn antigens, can be detected using the macrophage galactose-type C-type lectin (MGL/CLEC10A/CD301), which has been shown to be a promising tool for CRC prognosis. We had recently identified the major MGL binding glycoproteins in two high MGL binding CRC cells lines, HCT116 and HT29. We investigated here the impact of N-linked and O-linked glycans carried by these proteins for the binding to MGL. In addition, we performed quantitative proteomics to study the major differences in proteins involved in glycosylation in these cells. Our results showed that N-glycans have a significant, previously underestimated, importance in MGL binding to CRC cell lines.

2020-08-10 | PXD020344 | Pride

Novel Role for Macrophage Galactose-type Lectin-1 to Regulate Innate Immunity against Mycobacterium tuberculosis

Project description:Tuberculosis (TB) caused by infection with Mycobacterium tuberculosis (Mtb) is characterized by inflammatory pathology and poorly understood mechanisms of innate immunity. Pattern recognition receptors (PRR), expressed on the surface of macrophages, determine the balance of inflammatory and antimicrobial functions that influence disease outcome. Carbohydrate moieties displayed by mycobacteria can serve as PRR ligands for some members of the C-type lectin receptor (CLR) family; interactions that mediate a variety of incompletely understood immune outcomes. This work identifies a novel role for the CLR Macrophage Galactose-type Lectin-1 (MGL-1) in a mouse model of experimental tuberculosis. Murine macrophages upregulated MGL-1 following in vitro exposure to Mtb, while MGL+ cells accumulated at sites of mycobacteria-driven inflammation in the lung. Pulmonary macrophages from MGL-1-deficient mice displayed increased production of pro-inflammatory cytokines (IL-1b, IL-6 and IFN-g) that was associated with greater lipid accumulation, following Mtb infection. Surprisingly for a CLR, we also observed MGL-1-dependent anti-mycobacterial activity as evidenced by greater Mtb proliferation in BMDM, and the lung, of MGL-1 deficient mice. These results identify MGL-1 signaling as an important mechanism that regulates innate immunity against Mtb and indicate potential for the MGL pathway as a novel therapeutic target for anti-TB immunity.

2021-04-30 | GSE168486 | GEO

Simultaneously Identifying and Distinguishing Glycoproteins with O-GlcNAc and the Tn Antigen in Human Cancer Cells

Project description:Proteins modified with O-GlcNAc and O-GalNAc (the Tn antigen) play critically important roles in many physiological and pathological processes. Comprehensive and site-specific analysis of proteins modified with O-GlcNAc and the Tn antigen advances our understanding of glycoprotein functions and cellular activities. However, it is extraordinarily challenging to distinguish them because of the same chemical composition (identical molecular weights) and being localized on the same amino acid residues (S/T). Here, an effective method is developed through the integration of metabolic labeling, chemical and enzymatic reactions, and MS-based proteomics to simultaneously identify and distinguish glycoproteins modified with O-GlcNAc and the Tn antigen. In biologically duplicate experiments, 537 O-GlcNAcylated proteins and 103 glycoproteins modified with the Tn antigen were identified site-specifically in human cancer cells. Among O-GlcNAcylated proteins only in Jurkat cells, those involved in HTLV-1 infection were highly enriched, indicating that O-GlcNAcylation may play a role in regulating cellular response to the retrovirus. Glycoproteins in the spliceosome and with the nuclear localization signal (NLS) are also overrepresented. However, the O-GlcNAcylated proteins identified in all three cell lines were highly enriched with transcription and RNA binding functions. For glycoproteins with the Tn antigen, while many of them in Jurkat cells were highly enriched in the extracellular exosome, those in MCF7 and K562 cells were extensively found in the ER. The current method enables us to simultaneously analyze and unambiguously distinguish glycoproteins with O-GlcNAc and the Tn antigen, and it can be extensively applied for glycoscience research.

| MSV000088088 | MassIVE

EXoO-Tn: Tag-n-Map the Tn Antigen in the Human Proteome

Project description:Tn antigen (Tn), a single N-acetylgalactosamine (GalNAc) monosaccharide attached to protein Ser and Thr residues, is found on most solid tumors yet rarely detected in adult tissues: featuring it one of the most distinctive signatures of cancers. Although it is prevalent in cancers, Tn-glycosylation sites are not entirely clear owing to the lack of suitable technology. Knowing the Tn-glycosylation sites will spur the development of the new vaccines, diagnostics, and therapeutics of cancers. Here, we report a novel technology named EXoO-Tn for large-scale mapping of Tn-glycosylation sites. EXoO-Tn utilizes glycosyltransferase C1GalT1 and isotopically-labeled UDP-Gal(13C6) to tag and convert Tn to Gal(13C6)-Tn. This exquisite Gal(13C6)-Tn structure is recognized by a human-gut-bacterial enzyme, called OpeRATOR, that specifically cleaves N-termini of the Gal(13C6)-Tn-occupied Ser and Thr residues to yield site-containing glycopeptides. The enzymes C1GalT1 and OpeRATOR could be used concurrently in one-pot. The effectiveness of EXoO-Tn was evaluated by analyzing Jurkat cells, where 947 Tn-glycosylation sites from 480 glycoproteins were mapped. Bioinformatic analysis of the identified site-specific Tn-glycoproteins revealed conserved motif, cellular localization, relative position in proteins, and mapped site-specific Tn-glycoproteome in different studies. Given the significance of Tn in cancers, EXoO-Tn is anticipated to have broad utilities in clinical study of cancers.

2022-02-15 | PXD014390 | Pride

Large-scale and Site-specific Mapping of Intact O-glycans on Glycopeptides with IMPa

Project description:We employed the newly available IMPa O-glycoprotease from Pseudomonas aeruginosa for O-glycoproteomics analysis of cultured cells and tissues. The glycopeptides were extracted, purified, and conjugated to a solid support before an enzymatic cleavage by IMPa. O-glycopeptides were analyzed by EThcD, which allowed for a site-specific and global analysis of O-glycans, especially those sialylated and the Tn antigen. Through wild-type or SimpleCell HEK293 cells, we present two approaches, one for the analysis of total O-glycoproteome and the other for the Tn glycoproteome, and showed that IMPa and EThcD are necessary for confident localization of O-glycans on glycopeptides. We applied this method to study the O-glycoproteome of mouse brain, which revealed the high frequency of sialylated O-glycans and the Tn antigen on brain glycoproteins.

2023-08-28 | PXD037415 | Pride

Molecular Characterization of Basal-like and Non-Basal-like Triple-Negative Breast Cancers

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2014-07-29 | E-GEOD-41119 | biostudies-arrayexpress

Next generation sequencing of human monocyte derived dendritic cells stimulated with MGL-stimulating glycodendrimers in the absence or presence of LPS

Project description:To investigate the effect of MGL ligation in monocyte-derived dendritic cell biology, we generated control dendrimers and two different glycodendrimers exposing the MGL ligands αGalNAc or GalNAcβ1-4Gal. αGalNAc and GalNAcβ1-4Gal glycodendrimers were validated in the corresponding manuscript. Monocyte-derirved dendritic cells were generated by culturing monocytes for 4 days with IL-4 and GM-CSF. To study the effect of MGL-ligation at the transcriptional level, next generation sequencing was performed on monocyte-derived dendritic cells from three independent donors, stimulated for 4 h with control, αGalNAc or GalNAcβ1-4Gal glycodendrimers in the absence or presence of LPS.

2020-02-18 | GSE143699 | GEO

Transcriptional Response of Mycobacterium tuberculosis CDC1551 Rv0516c::Tn to treatment with 140 mM NaCl for 1 hr

Project description:To characterize the role of Rv0516c in the osmotic stress response of Mycobacterium tuberculosis (Mtb), we performed transcriptional profiling of CDC1551 Rv0516c::Tn following treatment with 140 mM NaCl for 1 hr relative to an untreated control. CDC1551 Rv0516c::Tn was treated with 140 mM NaCl for 1 h and compared to the same strain treated with 0 mM NaCl for 0 h. 4 biological replicates, independently grown and harvested. One replicate per array.

2013-11-15 | E-GEOD-50160 | biostudies-arrayexpress

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