N-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sda Synthase B4GALNT2.

The Sda carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human B4GALNT2 gene drives the expression of a long and a short protein isoform (LF-B4GALNT2 and SF-B4GALNT2) sharing identical transmembrane and luminal domains. Both isoforms are trans-Golgi proteins and the LF-B4GALNT2 also localizes to post-Golgi vesicles thanks to its extended cytoplasmic tail. Control mechanisms underpinning Sda and B4GALNT2 expression in the gastrointestinal tract are complex and not fully understood. This study reveals the existence of two unusual N-glycosylation sites in B4GALNT2 luminal domain. The first atypical N-X-C site is evolutionarily conserved and occupied by a complex-type N-glycan. We explored the influence of this N-glycan using site-directed mutagenesis and showed that each mutant had a slightly decreased expression level, impaired stability, and reduced enzyme activity. Furthermore, we observed that the mutant SF-B4GALNT2 was partially mislocalized in the endoplasmic reticulum, whereas the mutant LF-B4GALNT2 was still localized in the Golgi and post-Golgi vesicles. Lastly, we showed that the formation of homodimers was drastically impaired in the two mutated isoforms. An AlphaFold2 model of the LF-B4GALNT2 dimer with an N-glycan on each monomer corroborated these findings and suggested that N-glycosylation of each B4GALNT2 isoform controlled their biological activity.

Fluorescent GD2 analog for single-molecule imaging.

Ganglioside GD2 is associated with the proliferation and migration of breast cancer cells. However, the precise role of GD2 is unclear because its tendency to form dynamic and transient domains in cell plasma membranes (PMs), called lipid rafts, makes it difficult to observe. Previously, we developed fluorescent analogs of gangliosides (e.g., GM3 and GM1), which enabled the observation of lipid raft formation for the first time using single-molecule imaging. In this report, we describe the first chemical synthesis of a fluorescent ganglioside, GD2. A biophysical analysis of the synthesized analog revealed its raft-philic character, suggesting its potential to aid single-molecule imaging-based investigations into raft-associated interactions.

Analysis of the proximal promoter of the human colon-specific B4GALNT2 (Sda synthase) gene: B4GALNT2 is transcriptionally regulated by ETS1.

BACKGROUND: The Sda antigen and corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in normal colonic mucosa and are down-regulated to a variable degree in colon cancer tissues. Although their expression profile is well studied, little is known about the underlying regulatory mechanisms. METHODS: To clarify the molecular basis of Sda expression in the human gastrointestinal tract, we investigated the transcriptional regulation of the human B4GALNT2 gene. The proximal promoter region was delineated using luciferase assays and essential trans-acting factors were identified through transient overexpression and silencing of several transcription factors. RESULTS: A short cis-regulatory region restricted to the -72 to +12 area upstream of the B4GALNT2 short-type transcript variant contained the essential promoter activity that drives the expression of the human B4GALNT2 regardless of the cell type. We further showed that B4GALNT2 transcriptional activation mostly requires ETS1 and to a lesser extent SP1. CONCLUSIONS: Results presented herein are expected to provide clues to better understand B4GALNT2 regulatory mechanisms.

B4GALNT2 Controls Sda and SLex Antigen Biosynthesis in Healthy and Cancer Human Colon.

The Sda carbohydrate antigen and the corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in human normal colonic mucosa and are down-regulated to variable degrees in colon cancer. On the other hand, the tumor associated antigen SLex is not detected in the healthy colon and is upregulated in colon cancer. High level of B4GALNT2 gene expression appears to be a good marker of prognosis in colon cancer; however, the molecular mechanisms regulating these carbohydrate antigens' expression are still poorly understood. We review here the most recent progress made towards understanding this balanced expression of blood group carbohydrate epitopes Sda and SLex . In particular in recent years, we have attained a better understanding of genetic and epigenetic regulation of the B4GALNT2 gene and of the subcellular fate of B4GALNT2 isoforms.

Role of Sialyl-O-Acetyltransferase CASD1 on GD2 Ganglioside O-Acetylation in Breast Cancer Cells.

The O-acetylated form of GD2, almost exclusively expressed in cancerous tissues, is considered to be a promising therapeutic target for neuroectoderm-derived tumors, especially for breast cancer. Our recent data have shown that 9-O-acetylated GD2 (9-OAcGD2) is the major O-acetylated ganglioside species in breast cancer cells. In 2015, Baumann et al. proposed that Cas 1 domain containing 1 (CASD1), which is the only known human sialyl-O-acetyltransferase, plays a role in GD3 O-acetylation. However, the mechanisms of ganglioside O-acetylation remain poorly understood. The aim of this study was to determine the involvement of CASD1 in GD2 O-acetylation in breast cancer. The role of CASD1 in OAcGD2 synthesis was first demonstrated using wild type CHO and CHOΔCasd1 cells as cellular models. Overexpression using plasmid transfection and siRNA strategies was used to modulate CASD1 expression in SUM159PT breast cancer cell line. Our results showed that OAcGD2 expression was reduced in SUM159PT that was transiently depleted for CASD1 expression. Additionally, OAcGD2 expression was increased in SUM159PT cells transiently overexpressing CASD1. The modulation of CASD1 expression using transient transfection strategies provided interesting insights into the role of CASD1 in OAcGD2 and OAcGD3 biosynthesis, and it highlights the importance of further studies on O-acetylation mechanisms.

Cancer-Associated Glycosphingolipids as Tumor Markers and Targets for Cancer Immunotherapy.

Aberrant expression of glycosphingolipids is a hallmark of cancer cells and is associated with their malignant properties. Disialylated gangliosides GD2 and GD3 are considered as markers of neuroectoderm origin in tumors, whereas fucosyl-GM1 is expressed in very few normal tissues but overexpressed in a variety of cancers, especially in small cell lung carcinoma. These gangliosides are absent in most normal adult tissues, making them targets of interest in immuno-oncology. Passive and active immunotherapy strategies have been developed, and have shown promising results in clinical trials. In this review, we summarized the current knowledge on GD2, GD3, and fucosyl-GM1 expression in health and cancer, their biosynthesis pathways in the Golgi apparatus, and their biological roles. We described how their overexpression can affect intracellular signaling pathways, increasing the malignant phenotypes of cancer cells, including their metastatic potential and invasiveness. Finally, the different strategies used to target these tumor-associated gangliosides for immunotherapy were discussed, including the use and development of monoclonal antibodies, vaccines, immune system modulators, and immune effector-cell therapy, with a special focus on adoptive cellular therapy with T cells engineered to express chimeric antigen receptors.

[Reticular and Golgi glycosylation: Advances and associated diseases]. / Les mécanismes de régulation de la glycosylation - Exemples d'altérations des chaînes glycanniques dans les cancers.

Glycosylation is one of the essential modifications of proteins and lipids. It is carried out mainly in the endoplasmic reticulum and Golgi apparatus, and requires a specific molecular machinery associating several hundreds of glycosyltransferases, glycosidases, transporters and regulating proteins. Modifications of glycosylation are found in numerous diseases, notably in cancers. All types of glycosylation can be affected and this leads to dysfunctions of cellular metabolism. In this review, we present the current knowledge on the regulation of glycosylation mechanisms and illustrate how the alteration of these regulatory mechanisms can lead to abnormal protein and lipid glycosylation, and take part in the development of cancers.

TITLE: Les mécanismes de régulation de la glycosylation - Exemples d'altérations des chaînes glycanniques dans les cancers. ABSTRACT: La glycosylation est l'une des modifications essentielles des protéines et des lipides. Elle s'effectue principalement dans le réticulum endoplasmique et l'appareil de Golgi et fait appel à une machinerie moléculaire spécifique, associant plusieurs centaines de glycosyltransférases, de glycosidases, de transporteurs et de protéines régulatrices. Des modifications de la glycosylation sont retrouvées dans certaines maladies, notamment dans les cancers. Ces altérations peuvent affecter toutes les formes de glycosylation réticulaires et/ou golgiennes, et conduire à des dysfonctionnements du métabolisme cellulaire. Dans cette revue, nous présentons l'état actuel des connaissances des mécanismes de la glycosylation. Nous illustrerons, au travers d'exemples représentatifs, comment l'altération de certains de ces mécanismes de régulation peut affecter les différentes formes de glycosylation des protéines et des lipides et participer au développement des cancers.

Mycobacterium bovis BCG infection alters the macrophage N-glycome.

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-MSn glycotope-centric analysis of total N-glycans demonstrated that the increased fucosylation was due to an increased expression of the Lex (Galß1-4[Fucα1-3]GlcNAc) epitope, also known as stage-specific embryonic antigen-1. Modification of the surface expression of Lex 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.

O-acetylated Gangliosides as Targets for Cancer Immunotherapy.

O-acetylation of sialic acid residues is one of the main modifications of gangliosides, and modulates ganglioside functions. O-acetylation of gangliosides is dependent on sialyl-O-acetyltransferases and sialyl-O-acetyl-esterase activities. CAS1 Domain-Containing Protein 1 (CASD1) is the only human sialyl-O-acetyltransferases (SOAT) described until now. O-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 O-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 O-acetylation analysis and expression in cancers, and of the possible use of O-acetylated gangliosides as targets for cancer immunotherapy.

Glycosylation changes in inflammatory diseases.

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.

Profiling of O-acetylated Gangliosides Expressed in Neuroectoderm Derived Cells.

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 OAcGD2 expression in breast cancer (BC) cell lines. However, antibodies specific for O-acetylated gangliosides are not exempt of limitations, as they only provide information on the expression of a limited set of O-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 O-acetylated species that are usually lost under alkaline conditions and require specific analytical procedures. We used purification and extraction methods that preserve the O-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 O-acetylated gangliosides, we characterized the expression of OAcGM1, OAcGD3, OAcGD2, OAcGT2, and OAcGT3. Furthermore, the experimental procedure allowed us to clearly identify the position of the sialic acid residue that carries the O-acetyl group on b- and c-series gangliosides by MS/MS fragmentation. These results show that ganglioside O-acetylation occurs on both inner and terminal sialic acid residue in a cell type-dependent manner, suggesting different O-acetylation pathways for gangliosides. They also highlight the limitation of immuno-detection for the complete identification of O-acetylated ganglioside profiles in cancer cells.

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

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 O-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.

Identification of 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) as main O-acetylated sialic acid species of GD2 in breast cancer cells.

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 (Neu5,9Ac2). These results highlight a close interrelationship between Neu5,9Ac2 and OAcGD2 expression, and suggest that OAcGD2 is synthetized from GD2 and not from OAcGD3 in BC cells.

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

The Sda /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 ß1,4-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 ß1,4GalNAcT II, ß1,4-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.

Gangliosides in Cancer Cell Signaling.

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.

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

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.

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

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.

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

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, ß-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.

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

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-Lewisx (sLex) 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 sLex 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.

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

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.