Publications by authors named "Sophie Groux-Degroote"

27 Publications

  • Page 1 of 1

Mycobacterium bovis BCG infection alters the macrophage N-glycome.

Mol Omics 2020 08 9;16(4):345-354. Epub 2020 Apr 9.

Univ. Lille, CNRS UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, 59 000 Lille, France.

Macrophage glycosylation is essential to initiate the host-immune defense but may also be targeted by pathogens to promote infection. Indeed, the alteration of the cell-surface glycosylation status may affect the binding of lectins involved in cell activation and adhesion. Herein, we demonstrate that infection by M. bovis BCG induces the remodeling of the N-glycomes of both human primary blood monocyte-derived macrophages (MDM) and macrophage-cell line THP1. MALDI-MS based N-glycomic analysis established that mycobacterial infection induced increased synthesis of biantennary and multifucosylated complex type N-glycans. In contrast, infection of macrophages by M. bovis BCG did not modify the glycosphingolipids composition of macrophages. Further nano-LC-MS glycotope-centric analysis of total N-glycans demonstrated that the increased fucosylation was due to an increased expression of the Le (Galβ1-4[Fucα1-3]GlcNAc) epitope, also known as stage-specific embryonic antigen-1. Modification of the surface expression of Le was further confirmed in both MDM and THP-1 cells by FACS analysis using an α1,3-linked fucose specific lectin. Activation with the mycobacterial lipopeptide Pam3Lp19, an agonist of toll-like receptor 2, did not modify the overall fucosylation pattern, which suggests that the infection process is required to modify surface glycosylation. These results pave the way toward the understanding of infection-triggered cell-surface remodeling of macrophages.
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http://dx.doi.org/10.1039/c9mo00173eDOI Listing
August 2020

-acetylated Gangliosides as Targets for Cancer Immunotherapy.

Cells 2020 03 17;9(3). Epub 2020 Mar 17.

UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, CNRS, Université de Lille, F-59000 Lille, France.

-acetylation of sialic acid residues is one of the main modifications of gangliosides, and modulates ganglioside functions. -acetylation of gangliosides is dependent on sialyl--acetyltransferases and sialyl--acetyl-esterase activities. CAS1 Domain-Containing Protein 1 (CASD1) is the only human sialyl--acetyltransferases (SOAT) described until now. -acetylated ganglioside species are mainly expressed during embryonic development and in the central nervous system in healthy adults, but are re-expressed during cancer development and are considered as markers of cancers of neuroectodermal origin. However, the specific biological roles of -acetylated gangliosides in developing and malignant tissues have not been extensively studied, mostly because of the requirement of specific approaches and tools for sample preparation and analysis. In this review, we summarize our current knowledge of ganglioside biosynthesis and expression in normal and pathological conditions, of ganglioside -acetylation analysis and expression in cancers, and of the possible use of -acetylated gangliosides as targets for cancer immunotherapy.
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http://dx.doi.org/10.3390/cells9030741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140702PMC
March 2020

Glycosylation changes in inflammatory diseases.

Adv Protein Chem Struct Biol 2020 26;119:111-156. Epub 2019 Nov 26.

University Lille, CNRS, UMR 8576 - UGSF - Unite de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France.

Glycosylation is one of the most important modifications of proteins and lipids, and cell surface glycoconjugates are thought to play important roles in a variety of biological functions including cell-cell and cell-substrate interactions, bacterial adhesion, cell immunogenicity and cell signaling. Alterations of glycosylation are observed in a number of inflammatory diseases. Pro-inflammatory cytokines have been shown to modulate cell surface glycosylation by regulating the expression of glycosyltransferases and sulfotransferases involved in the biosynthesis of glycan chains, inducing the expression of specific carbohydrate antigens at the cell surface that can be recognized by different types of lectins or by bacterial adhesins, contributing to the development of diseases. Glycosylation can also regulate biological functions of immune cells by recruiting leukocytes to inflammation sites with pro- or anti-inflammatory effects. Cell surface proteoglycans provide a large panel of binding sites for many mediators of inflammation, and regulate their bio-availability and functions. In this review, we summarize the current knowledge of the glycosylation changes occurring in mucin type O-linked glycans, glycosaminoglycans, as well as in glycosphingolipids, with a particular focus on cystic fibrosis and neurodegenerative diseases, and their consequences on cell interactions and disease progression.
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http://dx.doi.org/10.1016/bs.apcsb.2019.08.008DOI Listing
January 2021

Profiling of -acetylated Gangliosides Expressed in Neuroectoderm Derived Cells.

Int J Mol Sci 2020 Jan 6;21(1). Epub 2020 Jan 6.

Univ. Lille, CNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France.

The expression and biological functions of oncofetal markers GD2 and GD3 were extensively studied in neuroectoderm-derived cancers in order to characterize their potential as therapeutic targets. Using immunological approaches, we previously identified GD3, GD2, and AcGD2 expression in breast cancer (BC) cell lines. However, antibodies specific for -acetylated gangliosides are not exempt of limitations, as they only provide information on the expression of a limited set of -acetylated ganglioside species. Consequently, the aim of the present study was to use structural approaches in order to apprehend ganglioside diversity in melanoma, neuroblastoma, and breast cancer cells, focusing on -acetylated species that are usually lost under alkaline conditions and require specific analytical procedures. We used purification and extraction methods that preserve the -acetyl modification for the analysis of native gangliosides by MALDI-TOF. We identified the expression of GM1, GM2, GM3, GD2, GD3, GT2, and GT3 in SK-Mel28 (melanoma), LAN-1 (neuroblastoma), Hs 578T, SUM 159PT, MDA-MB-231, MCF-7 (BC), and BC cell lines over-expressing GD3 synthase. Among -acetylated gangliosides, we characterized the expression of AcGM1, AcGD3, AcGD2, AcGT2, and AcGT3. Furthermore, the experimental procedure allowed us to clearly identify the position of the sialic acid residue that carries the -acetyl group on b- and c-series gangliosides by MS/MS fragmentation. These results show that ganglioside -acetylation occurs on both inner and terminal sialic acid residue in a cell type-dependent manner, suggesting different -acetylation pathways for gangliosides. They also highlight the limitation of immuno-detection for the complete identification of -acetylated ganglioside profiles in cancer cells.
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http://dx.doi.org/10.3390/ijms21010370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981417PMC
January 2020

Gangliosides: The Double-Edge Sword of Neuro-Ectodermal Derived Tumors.

Biomolecules 2019 07 27;9(8). Epub 2019 Jul 27.

Université de Lille, CNRS, UMR8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F59000 Lille, France.

Gangliosides, the glycosphingolipids carrying one or several sialic acid residues, are mostly localized at the plasma membrane in lipid raft domains and implicated in many cellular signaling pathways mostly by interacting with tyrosine kinase receptors. Gangliosides are divided into four series according to the number of sialic acid residues, which can be also modified by -acetylation. Both ganglioside expression and sialic acid modifications can be modified in pathological conditions such as cancer, which can induce either pro-cancerous or anti-cancerous effects. In this review, we summarize the specific functions of gangliosides in neuro-ectodermal derived tumors, and their roles in reprogramming the lipidomic profile of cell membrane occurring with the induction of epithelial-mesenchymal transition.
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http://dx.doi.org/10.3390/biom9080311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723632PMC
July 2019

Identification of 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac) as main O-acetylated sialic acid species of GD2 in breast cancer cells.

Glycoconj J 2019 02 5;36(1):79-90. Epub 2019 Jan 5.

CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, University Lille, F-59000, Lille, France.

Mainly restricted to the nervous system in healthy adults, complex gangliosides such as GD3 and GD2 have been shown to be involved in aggressiveness and metastasis of neuro-ectoderm derived tumors such as melanoma and neuroblastoma. Interestingly, O-acetylated forms of GD2, not expressed in human peripheral nerve fibers, are highly expressed in GD2+ tumor cells. Very little information is known regarding the expression of O-acetylated disialogangliosides in breast cancer (BC) cell lines. Here, we analyzed the expression of GD2, GD3 and their O-acetylated forms O-acetyl-GD2 (OAcGD2) and O-acetyl-GD3 (OAcGD3) in BC cells. We used Hs 578T and SUM159PT cell lines, as well as cell clones over-expressing GD3 synthase derived from MDA-MB-231 and MCF-7. Using flow cytometry and immunocytochemistry/confocal microscopy, we report that BC cells express b-series gangliosides GD3 and GD2, as well as significant amounts of OAcGD2. However, OAcGD3 expression was not detected in these cells. O-acetylation of gangliosides isolated from BC cells was examined by LC-MS analysis of sialic acid DMB-derivatives. We report that the main acetylated form of sialic acid expressed in BC gangliosides is 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac). These results highlight a close interrelationship between Neu5,9Ac and OAcGD2 expression, and suggest that OAcGD2 is synthetized from GD2 and not from OAcGD3 in BC cells.
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http://dx.doi.org/10.1007/s10719-018-09856-wDOI Listing
February 2019

The extended cytoplasmic tail of the human B4GALNT2 is critical for its Golgi targeting and post-Golgi sorting.

FEBS J 2018 09 31;285(18):3442-3463. Epub 2018 Aug 31.

Univ. Lille, CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France.

The Sd /Cad antigen reported on glycoconjugates of human tissues has an increasingly recognized wide impact on the physio-pathology of different biological systems. The last step of its biosynthesis relies on the enzymatic activity of the β1,4-N-acetylgalactosaminyltransferase-II (B4GALNT2), which shows the highest expression level in healthy colon. Previous studies reported the occurrence in human colonic cells of two B4GALNT2 protein isoforms that differ in the length of their cytoplasmic tail, the long isoform showing an extended 66-amino acid tail. We examined here, the subcellular distribution of the two B4GALNT2 protein isoforms in stably transfected colonic LS174T cells and in transiently transfected HeLa cells using fluorescence microscopy. While a similar subcellular distribution at the trans-Golgi cisternae level was observed for the two isoforms, our study pointed to an atypical subcellular localization of the long B4GALNT2 isoform into dynamic vesicles. We demonstrated a critical role of its extended cytoplasmic tail for its Golgi targeting and post-Golgi sorting and highlighted the existence of a newly described post-Golgi sorting signal as well as a previously undescribed fate of a Golgi glycosyltransferase.

Database: The proteins β1,4GalNAcT II, β1,4-GalT1, FucT I, FucT VI and ST3Gal IV are noted B4GALNT2, B4GALT1, FUT1, FUT6 and ST3GAL4, whereas the corresponding human genes are noted B4GALNT2, B4GALT1, FUT1, FUT6 and ST3GAL4 according to the HUGO nomenclature.
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http://dx.doi.org/10.1111/febs.14621DOI Listing
September 2018

Gangliosides in Cancer Cell Signaling.

Prog Mol Biol Transl Sci 2018 19;156:197-227. Epub 2018 Jan 19.

University of Lille, Villeneuve d'Ascq, France. Electronic address:

At the outer leaflet of the plasma membrane, gangliosides are found with other glycosphingolipids, phospholipids, and cholesterol in glycolipid-enriched microdomains, in which they interact with signaling molecules including receptor tyrosine kinases and signal transducers. The role of gangliosides in the regulation of signal transduction has been reported for many cases and in different cell types. The biosynthesis of gangliosides involves specific enzymes, mainly glycosyltransferases that control together with glycohydrolases, the steady state of gangliosides at the cell surface. Changes in ganglioside composition are therefore correlated with modifications of glycosyltransferases or glycohydrolases expression and result in the deregulation of cellular signals. In several types of cancers, the overexpression of disialogangliosides, such as GD3 or GD2 mainly results in the activation of cell signaling, increasing cell proliferation and migration, as well as tumor growth. In this chapter, we summarize our current knowledge of ganglioside biosynthesis, degradation, and of their role in cell signaling regulation in cancers.
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http://dx.doi.org/10.1016/bs.pmbts.2017.10.003DOI Listing
February 2019

TNF differentially regulates ganglioside biosynthesis and expression in breast cancer cell lines.

PLoS One 2018 26;13(4):e0196369. Epub 2018 Apr 26.

University of Lille, Structural and Functional Glycobiology Unit, UMR CNRS 8576, Lille, France.

Gangliosides are glycosphingolipids concentrated in glycolipid-enriched membrane microdomains. Mainly restricted to the nervous system in healthy adult, complex gangliosides such as GD3 and GD2 have been shown to be involved in aggressiveness and metastasis of neuro-ectoderm derived tumors such as melanoma and neuroblastoma. GD3 synthase (GD3S), the key enzyme that controls the biosynthesis of complex gangliosides, was shown to be over-expressed in Estrogen Receptor (ER)-negative breast cancer tumors, and associated with a decreased overall survival of patients. We previously demonstrated that GD3S expression in ER-negative breast cancer cells induced a proliferative phenotype and an increased tumor growth. In addition, our results clearly indicate that Tumor Necrosis Factor (TNF) induced GD3S over-expression in breast cancer cells via NFκB pathway. In this study, we analyzed the effect of TNF on ganglioside biosynthesis and expression in breast cancer cells from different molecular subtypes. We showed that TNF up-regulated the expression of GD3S in MCF-7 and Hs578T cells, whereas no change was observed for MDA-MB-231. We also showed that TNF induced an increased expression of complex gangliosides at the cell surface of a small proportion of MCF-7 cells. These results demonstrate that TNF differentially regulates gangliosides expression in breast cancer cell lines and establish a possible link between inflammation at the tumor site environment, expression of complex gangliosides and tumor development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0196369PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919650PMC
August 2018

Gangliosides: Structures, Biosynthesis, Analysis, and Roles in Cancer.

Chembiochem 2017 07 24;18(13):1146-1154. Epub 2017 Apr 24.

Université de Lille, CNRS, UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, 59000, Lille, France.

Gangliosides are acidic glycosphingolipids containing one or more sialic acid residues. They are essential compounds at the outer leaflet of the plasma membrane, where they interact with phospholipids, cholesterol, and transmembrane proteins, forming lipid rafts. They are involved in cell adhesion, proliferation, and recognition processes, as well as in the modulation of signal transduction pathways. These functions are mainly governed by the glycan moiety, and changes in the structures of gangliosides occur under pathological conditions, particularly in neuro-ectoderm-derived cancers. With the progress in mass spectrometry analysis of gangliosides, their role in cancer progression can be now investigated in more detail. In this review we summarize the current knowledge on the biosynthesis of gangliosides and their role in cancers, together with the recent development of cancer immunotherapy targeting gangliosides.
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http://dx.doi.org/10.1002/cbic.201600705DOI Listing
July 2017

Role of Cytokine-Induced Glycosylation Changes in Regulating Cell Interactions and Cell Signaling in Inflammatory Diseases and Cancer.

Cells 2016 Nov 29;5(4). Epub 2016 Nov 29.

Structural and Functional Glycobiology Unit, UMR CNRS 8576, University of Lille, Villeneuve d'Ascq 59655, France.

Glycosylation is one of the most important modifications of proteins and lipids, and cell surface glycoconjugates are thought to play important roles in a variety of biological functions including cell-cell and cell-substrate interactions, bacterial adhesion, cell immunogenicity and cell signaling. Alterations of glycosylation are observed in number of diseases such as cancer and chronic inflammation. In that context, pro-inflammatory cytokines have been shown to modulate cell surface glycosylation by regulating the expression of glycosyltransferases involved in the biosynthesis of carbohydrate chains. These changes in cell surface glycosylation are also known to regulate cell signaling and could contribute to disease pathogenesis. This review summarizes our current knowledge of the glycosylation changes induced by pro-inflammatory cytokines, with a particular focus on cancer and cystic fibrosis, and their consequences on cell interactions and signaling.
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http://dx.doi.org/10.3390/cells5040043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5187527PMC
November 2016

TNF up-regulates ST3GAL4 and sialyl-Lewisx expression in lung epithelial cells through an intronic ATF2-responsive element.

Biochem J 2017 01 7;474(1):65-78. Epub 2016 Nov 7.

Université Lille, CNRS, UMR 8576, Unité de Glycobiologie Structurale etFonctionnelle (UGSF), F-59000 Lille, France

We have previously shown that tumor necrosis factor (TNF) induced the up-regulation of the sialyltransferase gene ST3GAL4 (α2,3-sialyltransferase gene) BX transcript through mitogen- and stress-activated kinase 1/2 (MSK1/2), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. This up-regulation resulted in sialyl-Lewis (sLe) overexpression on high-molecular-weight glycoproteins in inflamed airway epithelium and increased the adhesion of Pseudomonas aeruginosa PAO1 and PAK strains to lung epithelial cells. In the present study, we describe a TNF-responsive element in an intronic region of the ST3GAL4 gene, whose TNF-dependent activity is repressed by ERK/p38 and MSK1/2 inhibitors. This TNF-responsive element contains potential binding sites for ETS1 and ATF2 transcription factors related to TNF signaling. We also show that ATF2 is involved in TNF responsiveness, as well as in TNF-induced ST3GAL4 BX transcript and sLe overexpression in A549 lung epithelial cells. Moreover, we show that TNF induces the binding of ATF2 to the TNF-responsive element. Altogether, these data suggest that ATF2 could be a potential target to prevent inflammation-induced P. aeruginosa binding in the lung of patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis.
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http://dx.doi.org/10.1042/BCJ20160602DOI Listing
January 2017

Glycosylation Changes Triggered by the Differentiation of Monocytic THP-1 Cell Line into Macrophages.

J Proteome Res 2017 01 8;16(1):156-169. Epub 2016 Jul 8.

Univ. Lille , CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, F 59000 Lille, France.

The human acute monocytic leukemia cell line THP-1 is widely used as an in vitro phagocytic cell model because it exhibits several immune properties similar to native monocyte-derived macrophages. In this study, we investigated the alteration of N- and O-linked glycans as well as glycosphingolipids, during THP-1 differentiation, combining mass spectrometry, flow cytometry, and quantitative real-time PCR. Mass spectrometry revealed that macrophage differentiation led to a marked upregulation of expression of GM3 ganglioside as well as an increase in complex-type structures, particularly triantennary glycans, occurring at the expense of high-mannose N-glycans. Moreover, we observed a slight decrease in the proportion of multifucosylated N-glycans and α2,6-sialylation. The uncovered changes in glycosylation correlated with variations of gene expression of relevant glycosyltransferases and glycosidases including sialyltransferases, β-N-acetylglucosaminyltransferases, fucosyltransferases, and neuraminidase. Furthermore, using flow cytometry and antibodies directed against glycan structures, we confirmed that the alteration of glycosylation occurs at the cell surface of THP-1 macrophage-like cells. Altogether, we established that macrophagic maturation of THP-1 induces dramatic modifications of the surface glycosylation pattern that may result in differential interaction of monocytic and macrophagic THP-1 with immune or bacterial lectins.
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http://dx.doi.org/10.1021/acs.jproteome.6b00161DOI Listing
January 2017

Structural Characterization of Mucin O-Glycosylation May Provide Important Information to Help Prevent Colorectal Tumor Recurrence.

Front Oncol 2015 8;5:217. Epub 2015 Oct 8.

Structural and Functional Glycobiology Unit, UMR CNRS 8576, University of Lille , Villeneuve d'Ascq , France.

Although colorectal cancer is a preventable and curable disease if early stage tumors are removed, it still represents the second cause of cancer-related death worldwide. Surgical resection is the only curative treatment but once operated the patient is either subjected to adjuvant chemotherapy or not, depending on the invasiveness of the cancer and risks of recurrence. In this context, we investigated, by mass spectrometry (MS), alterations in the repertoire of glycosylation of mucins from colorectal tumors of various stages, grades, and recurrence status. Tumors were also compared with their counterparts in resection margins from the same patients and with healthy controls. The obtained data showed an important decrease in the level of expression of sialylated core 3-based O-glycans in tumors correlated with an increase in sialylated core 1 structures. No correlation was established between stages of the tumor samples and mucin O-glycosylation. However, with the notable exception of sialyl Tn antigens, tumors with recurrence presented a milder alteration of glycosylation profile than tumors without recurrence. These results suggest that mucin O-glycans from tumors with recurrence might mimic a healthier physiological situation, hence deceiving the immune defense system.
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http://dx.doi.org/10.3389/fonc.2015.00217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4597131PMC
October 2015

Carbohydrate-to-carbohydrate interactions between α2,3-linked sialic acids on α2 integrin subunits and asialo-GM1 underlie the bone metastatic behaviour of LNCAP-derivative C4-2B prostate cancer cells.

Biosci Rep 2014 Sep 17;34(5). Epub 2014 Sep 17.

*School of Science, Technology and Engineering Management, St. Thomas University, 16401 NW 37th Avenue, Miami Gardens, FL 33054, USA.

Complex interplays among proteins, lipids and carbohydrates can alter the phenotype and are suggested to have a crucial role in tumour metastasis. Our previous studies indicated that a complex of the GSLs (glycosphingolipids), AsGM1 (asialo-GM1), which lacks α2,3-linked sialic acid, and α2β1 integrin receptors is responsible for the metastatic behaviour of C4-2B prostate cancer cells. Herein, we identified and addressed the functional significance of changes in sialylation during prostate cancer progression. We observed an increase in α2,3-linked sialic acid residues on α2 subunits of α2β1 integrin receptors, correlating with increased gene expression of α2,3-STs (sialyltransferases), particularly ST3GAL3. Cell surface α2,3-sialylation of α2 subunits was required for the integrin α2β1-dependent cell adhesion to collagen type I and the same α2,3-linked sialic acid residues on the integrin receptor were responsible for the interaction with the carbohydrate moiety of AsGM1, explaining the complex formation between AsGM1 and α2β1 integrin receptors. These results provide novel insights into the role of sialic acids in the organization and function of important membrane components in invasion and metastatic processes.
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http://dx.doi.org/10.1042/BSR20140096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4166120PMC
September 2014

B4GALNT2 gene expression controls the biosynthesis of Sda and sialyl Lewis X antigens in healthy and cancer human gastrointestinal tract.

Int J Biochem Cell Biol 2014 Aug 19;53:442-9. Epub 2014 Jun 19.

Structural and Functional Glycobiology Unit, UMR CNRS 8576, University Lille Nord de France, Lille1, 59655 Villeneuve d'Ascq, France. Electronic address:

The histo blood group carbohydrate Sd(a) antigen and its cognate biosynthetic enzyme B4GALNT2 show the highest level of expression in normal colon. Their dramatic down regulation previously observed in colon cancer tissues could play a role in the concomitant elevation of the selectin ligand sLe(x), involved in metastasis. However, down regulation of sLe(x) expression by B4GALNT2 has been so far demonstrated in vitro, but not in tissues. The human B4GALNT2 gene specifies at least two transcripts, diverging in the first exon, never studied in normal and cancer tissues. The long form contains a 253 nt exon 1L; the short form contains a 38 nt exon 1S. Using qPCR, we showed that cell lines and normal or cancerous colon, expressed almost exclusively the short form, while the long form was mainly expressed by the embryonic colon fibroblast cell line CCD112CoN. Immunochemistry approaches using colon cancer cells permanently expressing either B4GALNT2 cDNAs as controls, led to the observation of several protein isoforms in human normal and cancerous colon, and cell lines. We showed that tissues expressing B4GALNT2 protein isoforms were able to induce Sd(a) and to inhibit sLe(x) expression; both of which are expressed mainly on PNGase F-insensitive carbohydrate chains. Concomitant expression of B4GALNT2 and siRNA-mediated inhibition of FUT6, the major fucosyltransferase involved in sLe(x) synthesis in colon, resulted in a cumulative inhibition of sLe(x). In normal colon samples a significant relationship between sLe(x) expression and the ratio between FUT6/B4GALNT2 activities exists, demonstrating for the first time a role for B4GALNT2 in sLe(x) inhibition in vivo.
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http://dx.doi.org/10.1016/j.biocel.2014.06.009DOI Listing
August 2014

TNF induces the expression of the sialyltransferase ST3Gal IV in human bronchial mucosa via MSK1/2 protein kinases and increases FliD/sialyl-Lewis(x)-mediated adhesion of Pseudomonas aeruginosa.

Biochem J 2014 Jan;457(1):79-87

*Université Lille Nord de France, Lille 1, UGSF (Unité de Glycobiologie Structurale et Fonctionnelle), CNRS (Centre National de la Recherche Scientifique), UMR 8576, 59650 Villeneuve d'Ascq, France.

We have shown previously that the pro-inflammatory cytokine TNF (tumour necrosis factor) could drive sLe(x) (sialyl-Lewis(x)) biosynthesis through the up-regulation of the BX transcript isoform of the ST3GAL4 (ST3 β-galactoside α-2,3-sialyltransferase 4) sialyltransferase gene in lung epithelial cells and human bronchial mucosa. In the present study, we show that the TNF-induced up-regulation of the ST3GAL4 BX transcript is mediated by MSK1/2 (mitogen- and stress-activated kinase 1/2) through the ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) pathways, and increases sLe(x) expression on high-molecular-mass glycoproteins in inflamed airway epithelium. We also show that the TNF-induced sLe(x) expression increases the adhesion of the Pseudomonas aeruginosa PAO1 and PAK strains to lung epithelial cells in a FliD-dependent manner. These results suggest that ERK and p38 MAPK, and the downstream kinase MSK1/2, should be considered as potential targets to hamper inflammation, bronchial mucin glycosylation changes and P. aeruginosa binding in the lung of patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis.
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http://dx.doi.org/10.1042/BJ20130989DOI Listing
January 2014

TNF regulates sialyl-Lewisx and 6-sulfo-sialyl-Lewisx expression in human lung through up-regulation of ST3GAL4 transcript isoform BX.

Biochimie 2012 Sep 9;94(9):2045-53. Epub 2012 Jun 9.

Université Lille Nord de France, Lille 1, UGSF, CNRS, UMR 8576, 59650 Villeneuve d'Ascq, France.

Bronchial mucins from severely infected patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis exhibit increased amounts of sialyl-Lewis(x) (NeuAcα2-3Galβ1-4[Fucα1-3]GlcNAc-R, sLe(x)) glycan structures. In cystic fibrosis, sLe(x) and its sulfated form 6-sulfo-sialyl-Lewis(x) (NeuAcα2-3Galβ1-4[Fucα1-3](HO(3)S-6)GlcNAc-R, 6-sulfo-sLe(x)) serve as receptors for Pseudomonas aeruginosa and are involved in the chronicity of airway infection. However, little is known about the molecular mechanisms regulating the changes in glycosylation and sulfation of mucins in airways. Herein, we show that the pro-inflammatory cytokine TNF increases the expression of α2,3-sialyltransferase gene ST3GAL4, both in human bronchial mucosa and in A549 lung carcinoma cells. The role of sialyltransferase ST3Gal IV in sLe(x) biosynthesis was confirmed by siRNA silencing of ST3GAL4 gene. BX is the major transcript isoform expressed in healthy bronchial mucosa and in A549 cells, and is up-regulated by TNF in both models. Bioinformatics analysis and luciferase assays have confirmed that the 2 kb genomic sequence surrounding BX exon contains a promoter region regulated by TNF-related transcription factors. These results support further work aiming at the development of anti-inflammatory strategy to reduce chronic airway infection in diseases such as cystic fibrosis.
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http://dx.doi.org/10.1016/j.biochi.2012.05.030DOI Listing
September 2012

Sialyltransferases functions in cancers.

Front Biosci (Elite Ed) 2012 Jan 1;4:499-515. Epub 2012 Jan 1.

Unite de Glycobiologie Structurale et Fonctionnelle, Universite Lille Nord de France, Lille1, CNRS UMR 8576, F-59655 Villeneuve d'Ascq, France.

Abnormally elevated levels of sialylated tumor associated carbohydrate antigens are frequently described at the surface of cancer cells and/or secreted in biological fluids. It is now well established that this over-expression may result from deregulation in sialyltransferases enzymatic activity involved in their biosynthesis, but the precise molecular mechanisms remain unknown. Twenty different human sialyltransferases preside to the sialylation of glycoconjugates, either glycolipids or glycoproteins. This review summarizes the current knowledge on human sialyltransferases implicated in the altered expression of sialylated tumor associated antigens, the molecular basis of their regulated expression in cancer cells and the various tools developed by researchers and clinicians for their study in pathological samples.
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http://dx.doi.org/10.2741/396DOI Listing
January 2012

Hyperacidification of trans-Golgi network and endo/lysosomes in melanocytes by glucosylceramide-dependent V-ATPase activity.

Traffic 2011 Nov 30;12(11):1634-47. Epub 2011 Aug 30.

Membrane Enzymology, Bijvoet Center and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands.

Sphingolipids are considered to play a key role in protein sorting and membrane trafficking. In melanocytic cells, sorting of lysosomal and melanosomal proteins requires the sphingolipid glucosylceramide (GlcCer). This sorting information is located in the lumenal domain of melanosomal proteins. We found that two processes dependent on lumenal pH, protein sialylation and lysosomal acid lipase (LAL) activity were aberrant in GM95 melanocyte cells, which do not produce glycosphingolipids. Using fluorescence lifetime imaging microscopy (FLIM), we found that the lumenal pH in the trans-Golgi network and lysosomes of wild-type melanocyte MEB4 cells are >1 pH unit lower than GM95 cells and fibroblasts. In addition to the lower pH found in vivo, the in vitro activity of the proton pump, the vacuolar-type H(+) -translocating ATPase (V-ATPase), was twofold higher in MEB4 compared to GM95 cells. The apparent K(i) for inhibition of the V-ATPase by concanamycin A and archazolid A, which share a common binding site on the c-ring, was lower in glycosphingolipid-deficient GM95 cells. No difference between the MEB4 and GM95 cells was found for the V-ATPase inhibitors apicularen A and salicylihalimide. We conclude that hyperacidification in MEB4 cells requires glycosphingolipids and propose that low pH is necessary for protein sorting and melanosome biogenesis. Furthermore, we suggest that glycosphingolipids are indirectly involved in protein sorting and melanosome biogenesis by stimulating the proton pump, possibly through binding of GlcCer. These experiments establish, for the first time, a link between pH, glycosphingolipids and melanosome biogenesis in melanocytic MEB4 cells, to suggest a role for glycosphingolipids in hyperacidification in melanocytes.
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http://dx.doi.org/10.1111/j.1600-0854.2011.01263.xDOI Listing
November 2011

Consequences of the expression of sialylated antigens in breast cancer.

Carbohydr Res 2010 Jul 4;345(10):1377-83. Epub 2010 Feb 4.

Univ. Lille Nord de France, F-59000 Lille, France.

Changes in cell surface glycosylation are common modifications that occur during oncogenesis, leading to the over-expression of tumour-associated carbohydrate antigens (TACA). Most of these antigens are sialylated and the increase of sialylation is a well-known feature of transformed cells. In breast cancer, expression of TACA such as sialyl-Lewis(x) or sialyl-Tn is usually associated with a poor prognosis and a decreased overall survival of patients. However, the specific role of these sialylated antigens in breast tumour development and aggressiveness is not clearly understood. These glycosylation changes result from the modification of the expression of genes encoding specific glycosyltransferases involved in glycan biosynthesis and the level of expression of sialyltransferase genes has been proposed to be a prognostic marker for the follow-up of breast cancer patients. Several human cellular models have been developed in order to explain the mechanisms by which carbohydrate antigens can reinforce breast cancer progression and aggressiveness. TACA expression is associated with changes in cell adhesion, migration, proliferation and tumour growth. In addition, recent data on glycolipid biosynthesis indicate an important role of G(D3) synthase expression in breast cancer progression. The aim of this review is to summarize our current knowledge of sialylation changes that occur in breast cancer and to describe the cellular models developed to analyze the consequences of these changes on disease progression and aggressiveness.
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http://dx.doi.org/10.1016/j.carres.2010.01.024DOI Listing
July 2010

Transcriptional regulation of the human ST6GAL2 gene in cerebral cortex and neuronal cells.

Glycoconj J 2010 Jan 19;27(1):99-114. Epub 2009 Sep 19.

Structural and Functional Glycobiology Unit, UMR CNRS 8576, University of Sciences and Technologies of Lille, 59655, Villeneuve d'Ascq, France.

The second human beta-galactoside alpha-2,6-sialyltransferase (hST6Gal II) differs from hST6Gal I, the first member of ST6Gal family, in substrate specificity and tissue expression pattern. While ST6GAL1 gene is expressed in almost all human tissues, ST6GAL2 shows a restricted tissue-specific pattern of expression, mostly expressed in embryonic and adult brain. In order to understand the mechanisms involved in the transcriptional regulation of ST6GAL2, we first characterized the transcription start sites (TSS) in SH-SY5Y neuroblastoma cells. 5' RACE experiments revealed multiple TSS located on three first alternative 5' exons, termed EX, EY and EZ, which are unusually close on the genomic sequence and are all located more than 42 kbp upstream of the first common coding exon. Using Taqman duplex Q-PCR, we showed that the ST6GAL2 transcripts initiated by EX or EY are mainly expressed in both brain-related cell lines and human cerebral cortex, testifying for the use of a similar transcriptional regulation in vivo. Furthermore, we also showed for the first time hST6Gal II protein expression in the different lobes of the human cortex. Luciferase reporter assays allowed us to define two sequences upstream EX and EY with a high and moderate promoter activity, respectively. Bioinformatics analysis and site-directed mutagenesis showed that NF-kappaB and NRSF are likely to act as transcriptional repressors, whereas neuronal-related development factors Sox5, Puralpha and Olf1, are likely to act as transcriptional activators of ST6GAL2. This suggests that ST6GAL2 transcription could be potentially activated for specific neuronal functions.
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http://dx.doi.org/10.1007/s10719-009-9260-yDOI Listing
January 2010

Glycosyltransferase and sulfotransferase gene expression profiles in human monocytes, dendritic cells and macrophages.

Glycoconj J 2009 Dec;26(9):1259-74

Inserm, U547, Lille, 59019, France.

Using a focused glycan-gene microarray, we compared the glycosyltransferase (GT) and sulfotransferase gene expression profiles of human monocytes, dendritic cells (DCs) and macrophages (Mphis), isolated or differentiated from the same donors. Microarray analysis indicated that monocytes express transcripts for a full set of enzymes involved in the biosynthesis of multi-multiantennary branched N-glycans, potentially elongated by poly-N-acetyl-lactosamine chains, and of mucin-type Core 1 and Core 2 sialylated O-glycans. Monocytes also express genes involved in the biosynthesis and modification of glycosaminoglycans, but display a limited expression of GTs implicated in glycolipid synthesis. Among genes expressed in monocytes (90 out of 175), one third is significantly modulated in DCs and Mphi respectively, most of them being increased in both cell types relative to monocytes. These changes might potentially enforce the capacity of differentiated cells to synthesize branched N-glycans and mucin-type O-glycans and to remodel cell surface proteoglycans. Stimulation of DCs and Mphis with lipopolysaccharide caused a general decrease in gene expression, mainly affecting genes found to be positively modulated during the differentiation steps. Interestingly, although a similar set of enzymes are modulated in the same direction in mature DCs and Mphis, cell specific genes are also differentially regulated during maturation, a phenomenon that may sustain functional specificities. Validation of this analysis was provided by quantitative real-time PCR and flow cytometry of cell surface glycan antigens. Collectively, this study implies an important modification of the pattern of glycosylation in DCs and Mphis undergoing differentiation and maturation with potential biological consequences.
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http://dx.doi.org/10.1007/s10719-009-9244-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2967374PMC
December 2009

GD3 synthase overexpression enhances proliferation and migration of MDA-MB-231 breast cancer cells.

Biol Chem 2009 Jul;390(7):601-9

Structural and Functional Glycobiology Unit, UMR CNRS 8576, University of Sciences and Technologies of Lille, F-59655 Villeneuve d'Ascq, France.

The disialoganglioside G(D3) is an oncofetal marker of a variety of human tumors including melanoma and neuroblastoma, playing a key role in tumor progression. G(D3) and 9-O-acetyl-G(D3) are overexpressed in approximately 50% of invasive ductal breast carcinoma, but no relationship has been established between disialoganglioside expression and breast cancer progression. In order to determine the effect of G(D3) expression on breast cancer development, we analyzed the biosynthesis of gangliosides in several breast epithelial cell lines including MDA-MB-231, MCF-7, BT-20, T47-D, and MCF10A, by immunocytochemistry, flow cytometry, and real-time PCR. Our results show that, in comparison to tumors, cultured breast cancer cells express a limited pattern of gangliosides. Disialogangliosides were not detected in any cell line and G(M3) was only observed at the cell surface of MDA-MB-231 cells. To evaluate the influence of G(D3) in breast cancer cell behavior, we established and characterized MDA-MB-231 cells overexpressing G(D3) synthase. We show that G(D3) synthase expressing cells accumulate G(D3), G(D2), and G(T3) at the cell surface. Moreover, G(D3) synthase overexpression bypasses the need of serum for cell growth and increases cell migration. This suggests that G(D3) synthase overexpression may contribute to increasing the malignant properties of breast cancer cells.
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http://dx.doi.org/10.1515/BC.2009.054DOI Listing
July 2009

Glycolipid-dependent sorting of melanosomal from lysosomal membrane proteins by lumenal determinants.

Traffic 2008 Jun 28;9(6):951-63. Epub 2008 Mar 28.

Membrane Enzymology, Bijvoet Center and Institute of Biomembranes, Utrecht University, 3584CH Utrecht, The Netherlands.

Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal enzymes tyrosinase-related protein 1 (Tyrp1) and tyrosinase follow an intracellular Golgi to melanosome pathway, whereas in the absence of glycosphingolipids, they are observed to pass over the cell surface. Unexpectedly, the lysosome-associated membrane protein 1 (LAMP-1) and 2 behaved exactly opposite: they were found to travel through the cell surface in control melanocytes but followed an intracellular pathway in the absence of glycosphingolipids. Chimeric proteins having the cytoplasmic tail of Tyrp1 or tyrosinase were transported like lysosomal proteins, whereas a LAMP-1 construct containing the lumenal domain of Tyrp1 localized to melanosomes. In conclusion, the lumenal domain contains sorting information that guides Tyrp1 and probably tyrosinase to melanosomes by an intracellular route that excludes lysosomal proteins and requires glucosylceramide.
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http://dx.doi.org/10.1111/j.1600-0854.2008.00740.xDOI Listing
June 2008

IL-6 and IL-8 increase the expression of glycosyltransferases and sulfotransferases involved in the biosynthesis of sialylated and/or sulfated Lewisx epitopes in the human bronchial mucosa.

Biochem J 2008 Feb;410(1):213-23

Unité de Glycobiologie Structurale et Fonctionnelle, UMR CNRS No. 8576, Bâtiment C9, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq, Lille, France.

Bronchial mucins from patients suffering from CF (cystic fibrosis) exhibit glycosylation alterations, especially increased amounts of the sialyl-Lewis(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc-R) and 6-sulfo-sialyl-Lewis(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3][SO(3)H-6]GlcNAc-R) terminal structures. These epitopes are preferential receptors for Pseudomonas aeruginosa, the bacteria responsible for the chronicity of airway infection and involved in the morbidity and early death of CF patients. However, these glycosylation changes cannot be directly linked to defects in CFTR (CF transmembrane conductance regulator) gene expression since cells that secrete airway mucins express no or very low amounts of the protein. Several studies have shown that inflammation may affect glycosylation and sulfation of various glycoproteins, including mucins. In the present study, we show that incubation of macroscopically healthy fragments of human bronchial mucosa with IL-6 (interleukin-6) or IL-8 results in a significant increase in the expression of alpha1,3/4-fucosyltransferases [FUT11 (fucosyltransferase 11 gene) and FUT3], alpha2-6- and alpha2,3-sialyltransferases [ST3GAL6 (alpha2,3-sialyltransferase 6 gene) and ST6GAL2 (alpha2,6-sialyltransferase 2 gene)] and GlcNAc-6-O-sulfotransferases [CHST4 (carbohydrate sulfotransferase 4 gene) and CHST6] mRNA. In parallel, the amounts of sialyl-Lewis(x) and 6-sulfo-sialyl-Lewis(x) epitopes at the periphery of high-molecular-mass proteins, including MUC4, were also increased. In conclusion, our results indicate that IL-6 and -8 may contribute to the increased levels of sialyl-Lewis(x) and 6-sulfo-sialyl-Lewis(x) epitopes on human airway mucins from patients with CF.
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http://dx.doi.org/10.1042/BJ20070958DOI Listing
February 2008

Transcription factor AP-2alpha represses both the mucin MUC4 expression and pancreatic cancer cell proliferation.

Carcinogenesis 2007 Nov 9;28(11):2305-12. Epub 2007 Jul 9.

INSERM, U837, Place de Verdun, 59045 Lille cedex, France.

MUC4 is a transmembrane mucin expressed in pancreatic ductal adenocarcinoma (DAC) in contrast to normal pancreas, and is an independent predictor of poor prognosis in patients with invasive DAC. Our aim was therefore to investigate the mechanisms that control MUC4 expression in pancreatic cancer cells. We focused our study on activator protein (AP)-2alpha transcription factor that acts as a tumour suppressor gene in several cancers. In a series of 18 human DAC, using immunohistochemistry, we confirmed that MUC4 was exclusively expressed in cancerous or preneoplastic lesions in 83% of the samples. On the contrary, AP-2 was mainly expressed by non-tumoural ductal cells (61%) or endocrine cells (67%). Moreover, MUC4 and AP-2 were never found co-expressed suggesting an inhibitory role of AP-2alpha in normal ductal cells. In CAPAN-1 and CAPAN-2 cells, transient AP-2alpha over-expression decreased both MUC4 mRNA and apomucin levels by 20-40% by a mechanism involving inhibition of MUC4 promoter. By chromatin immunoprecipitation and gel-shift assays, we demonstrated that this inhibition involved two AP-2 cis-elements located in the -475/-238 region of the promoter. CAPAN-1 clones, which stably over-expressed AP-2alpha, displayed a strong MUC4 down-regulation (-38 to -100%), a significant decrease of both cell proliferation and invasion concomitant to the up-regulation of p27 cyclin-dependent kinase inhibitor. In conclusion, our data provide evidence that AP-2alpha is an important in vivo negative regulator of MUC4 expression in human pancreatic tissue and that AP-2alpha may play a tumour-suppressive role in pancreatic DAC.
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http://dx.doi.org/10.1093/carcin/bgm158DOI Listing
November 2007