Alterations in the glycomic profile are a hallmark of cancer, including colorectal cancer (CRC). While, the glycosylation of glycoproteins and glycolipids has been widely studied for CRC cell lines ...and tissues, a comprehensive overview of CRC glycomics is still lacking due to the usage of different samples and analytical methods. In this study, we compared glycosylation features of N-, O-glycans, and glycosphingolipid glycans for a set of 22 CRC cell lines, all measured by porous graphitized carbon nano-liquid chromatography-tandem mass spectrometry. An overall, high abundance of (sialyl)Lewis antigens for colon-like cell lines was found, while undifferentiated cell lines showed high expression of H blood group antigens and α2-3/6 sialylation. Moreover, significant associations of glycosylation features were found between the three classes of glycans, such as (sialyl)Lewis and H blood group antigens. Integration of the datasets with transcriptomics data revealed positive correlations between (sialyl)Lewis antigens, the corresponding glycosyltransferase FUT3 and transcription factors CDX1, ETS, HNF1/4A, MECOM, and MYB. This indicates a possible role of these transcription factors in the upregulation of (sialyl)Lewis antigens, particularly on glycosphingolipid glycans, via FUT3/4 expression in colon-like cell lines. In conclusion, our study provides insights into the possible regulation of glycans in CRC and can serve as a guide for the development of diagnostic and therapeutic biomarkers.
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•Glycosylation motifs are compared for a set of 22 colorectal cancer cell lines.•For (sialyl)Lewis and H blood group antigens significant associations are found across the three glycan classes.•Colon-like cell lines present high abundance of (sialyl)Lewis antigens.•High expression of (sialyl)Lewis antigens might result from the regulation of FUT3.
An integrated analysis of glycosylation features including (sialyl)Lewis structure and blood group antigens was performed across N-, O-glycans, and glycosphingolipid glycans. Colon-like lines exhibited abundant (sialyl)Lewis antigens, while undifferentiated lines expressed H blood group antigens and α2-3/6 sialylation. The observed associations across glycan classes along with correlations with glycosyltransferases and transcription factors suggest that certain transcription factors like CDX1 contribute to the upregulation of (sialyl)Lewis antigens on all three glycan classes via regulation of glycosyltransferases FUT3/4.
Until recently, glycan epitopes have not been documented by the WHO/IUIS Allergen Nomenclature Sub‐Committee. This was in part due to scarce or incomplete information on these oligosaccharides, but ...also due to the widely held opinion that IgE to these epitopes had little or no relevance to allergic symptoms. Most IgE‐binding glycans recognized up to 2008 were considered to be “classical” cross‐reactive carbohydrate determinants (CCD) that occur in insects, some helminths and throughout the plant kingdom. Since 2008, the prevailing opinion on lack of clinical relevance of IgE‐binding glycans has been subject to a reevaluation. This was because IgE specific for the mammalian disaccharide galactose‐alpha‐1,3‐galactose (alpha‐gal) was identified as a cause of delayed anaphylaxis to mammalian meat in the United States, an observation that has been confirmed by allergists in many parts of the world. Several experimental studies have shown that oligosaccharides with one or more terminal alpha‐gal epitopes can be attached as a hapten to many different mammalian proteins or lipids. The classical CCDs also behave like haptens since they can be expressed on proteins from multiple species. This is the explanation for extensive in vitro cross‐reactivity related to CCDs. Because of these developments, the Allergen Nomenclature Sub‐Committee recently decided to include glycans as potentially allergenic epitopes in an adjunct section of its website (www.allergen.org). In this article, the features of the main glycan groups known to be involved in IgE recognition are revisited, and their characteristic structural, functional, and clinical features are discussed.
Glycosylation is crucial in cellular metabolism and survival. Of interest is the role of N-linked and O-linked glycans in disease states. Robust analytical methods must be defined to identify ...suitable glycan biomarkers and glyco-therapeutics. Fortunately, in N-glycan analysis, a universal enzyme exists to deglycosylate a variety of common-core structures from proteins for analysis using mass spectrometric and fluorescence techniques. Unfortunately, for their O-linked counterparts, no such enzyme exists. Furthermore, O-glycan heterogeneity is vast due to the lack of a common glycan core, making analysis challenging. As such, chemical methods are used to liberate O-glycans, however, often to the detriment of the glycan’s structure due to “peeling” reactions. This review outlines approaches for O-glycan release and downstream glycomic and glycoproteomic analysis.
The O‐glycan branching enzyme, core2 β‐1,6‐N‐acetylglucosaminyltransferase (C2GnT), forms O‐glycans containing an N‐acetylglucosamine branch connected to N‐acetylgalactosamine (core2 O‐glycans) on ...cell‐surface glycoproteins. Here, we report that upregulation of C2GnT is closely correlated with progression of bladder tumours and that C2GnT‐expressing bladder tumours use a novel strategy to increase their metastatic potential. Our results showed that C2GnT‐expressing bladder tumour cells are highly metastatic due to their high ability to evade NK cell immunity and revealed the molecular mechanism of the immune evasion by C2GnT expression. Engagement of an NK‐activating receptor, NKG2D, by its tumour‐associated ligand, Major histocompatibility complex class I‐related chain A (MICA), is critical to tumour rejection by NK cells. In C2GnT‐expressing bladder tumour cells, poly‐N‐acetyllactosamine was present on core2 O‐glycans on MICA, and galectin‐3 bound the NKG2D‐binding site of MICA through this poly‐N‐acetyllactosamine. Galectin‐3 reduced the affinity of MICA for NKG2D, thereby severely impairing NK cell activation and silencing the NK cells. This new mode of NK cell silencing promotes immune evasion of C2GnT‐expressing bladder tumour cells, resulting in tumour metastasis.
This paper illuminates a new mechanism for tumour cell evasion from the immune surveillance. Molecular analyses reveal modifications of the NKG2D‐NKG2DL system that prevent natural killer cell activation.
Human immunodeficiency virus 1 (HIV‐1) virus‐like particles (VLPs) are nanostructures derived from the self‐assembly and cell budding of Gag polyprotein. Mimicking the native structure of the virus ...and being noninfectious, they represent promising candidates for the development of new vaccines as they elicit a strong immune response. In addition to this, the bounding membrane can be functionalized with exogenous antigens to target different diseases. Protein glycosylation depends strictly on the production platform and expression system used and the displayed glycosylation patterns may influence downstream processing as well as the immune response. One of the main challenges for the development of Gag VLP production bioprocess is the separation of VLPs and coproduced extracellular vesicles (EVs). In this study, porous graphitized carbon separation method coupled with mass spectrometry was used to characterize the N‐ and O‐ glycosylation profiles of Gag VLPs produced in HEK293 cells. We identified differential glycan signatures between VLPs and EVs that could pave the way for further separation and purification strategies to optimize downstream processing and move forward in VLP‐based vaccine production technology.
A detailed characterization of the different N‐ and O‐glycans present in the membrane of human immunodeficiency virus 1 (HIV‐1) Gag virus‐like particles (VLPs) and coproduced extracellular vesicles (EVs) in HEK293 was performed in this study. Significant changes in relative abundance of the glycan species between EVs and VLPs were identified, laying the groundwork for the future design of downstream separation strategies.
Abstract
Mucin-type O-glycans decorate >80% of secretory and cell surface proteins and contribute to health and disease. However, dynamic alterations in the O-glycome are poorly understood because ...current O-glycomic methodologies are not sufficiently sensitive nor quantitative. Here we describe a novel isotope labeling approach termed Isotope-Cellular O-glycome Reporter Amplification (ICORA) to amplify and analyze the O-glycome from cells. In this approach, cells are incubated with Ac3GalNAc-Bn (Ac3GalNAc-1H7Bn) or a heavy labeled Ac3GalNAc-BnD7 (Ac3GalNAc-2D7Bn) O-glycan precursor (7 Da mass difference), which enters cells and upon de-esterification is modified by Golgi enzymes to generate Bn-O-glycans secreted into the culture media. After recovery, heavy and light Bn-O-glycans from two separate conditions are mixed, analyzed by MS, and statistically interrogated for changes in O-glycan abundance using a semi-automated approach. ICORA enables ~100–1000-fold enhanced sensitivity and increased throughput compared to traditional O-glycomics. We validated ICORA with model cell lines and used it to define alterations in the O-glycome in colorectal cancer. ICORA is a useful tool to explore the dynamic regulation of the O-glycome in health and disease.
O-glycosylation is a highly frequent post-translation modification of proteins, with important functional implications in both physiological and disease contexts. The biosynthesis of O-glycans ...depends on several layers of regulation of the cellular glycosylation machinery, being organ-, tissue- and cell-specific. This review provides insights on the molecular mechanism underlying O-glycan biosynthesis and modification, and highlights illustrative examples of diseases that are triggered or modulated by aberrant cellular O-glycosylation. Particular relevance is given to genetic disorders of glycosylation, infectious diseases and cancer. Finally, we address the potential of O-glycans and their biosynthetic pathways as targets for novel therapeutic strategies.
•O-glycosylation is a highly regulated process with key roles in health and disease.•Defects affecting the O-glycan biosynthetic machinery impact several human diseases.•O-glycans are key modulators of host-pathogen interplay and immune response.•Aberrant cellular O-glycosylation promotes oncogenic signaling and tumor progression.•O-glycans represent promising molecular targets for innovative therapeutic approaches.
A complex mucus network made up of large polymers of the mucin-family glycoprotein MUC2 exists between the large intestinal microbial mass and epithelial and immune cells. This has long been ...understood as an innate immune defense barrier against the microbiota and other luminal threats that reinforces the barrier function of the epithelium and limits microbiota contact with the tissues. However, past and recent studies have provided new evidence of how critical the mucus network is to act as a ‘liaison’ between host and microbe to mediate anti-inflammatory, mutualistic interactions with the microbiota and protection from pathogens. This review summarizes historical and recent insights into the formation of the gut mucus network, how the microbes and immune system influence mucus, and in turn, how the mucus influences immune responses to the microbiota.
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Aberrant expression of Sialyl‐Tn (STn) antigen correlates with poor prognosis and reduced patient survival. We demonstrated that expression of Tn and STn in pancreatic ductal adenocarcinoma (PDAC) is ...due to hypermethylation of Core 1 synthase specific molecular chaperone (COSMC) and enhanced the malignant properties of PDAC cells with an unknown mechanism. To explore the mechanism, we have genetically deleted COSMC in PDAC cells to express truncated O‐glycans (SimpleCells, SC) which enhanced cell migration and invasion. Since epithelial‐to‐mesenchymal transition (EMT) play a vital role in metastasis, we have analysed the induction of EMT in SC cells. Expressions of the mesenchymal markers were significantly high in SC cells as compared to WT cells. Equally, we found reduced expressions of the epithelial markers in SC cells. Re‐expression of COSMC in SC cells reversed the induction of EMT. In addition to this, we also observed an increased cancer stem cell population in SC cells. Furthermore, orthotopic implantation of T3M4 SC cells into athymic nude mice resulted in significantly larger tumours and reduced animal survival. Altogether, these results suggest that aberrant expression of truncated O‐glycans in PDAC cells enhances the tumour aggressiveness through the induction of EMT and stemness properties.
Addition of sialic acids (sialylation) to glycoconjugates is a common capping step of glycosylation. Our study aims to determine the roles of the overall sialylation in intestinal mucosal ...homeostasis.
Mice with constitutive deletion of intestinal epithelial sialylation (IEC Slc35a1–/– mice) and mice with inducible deletion of sialylation in intestinal epithelium (TM-IEC Slc35a1–/– mice) were generated, which were used to determine the roles of overall sialylation in intestinal mucosal homeostasis by ex vivo and muti-omics studies.
IEC Slc35a1–/– mice developed mild spontaneous microbiota-dependent colitis. Additionally, 30% of IEC Slc35a1–/– mice had spontaneous tumors in the rectum over the age of 12 months. TM-IEC Slc35a1–/– mice were highly susceptible to acute inflammation induced by 1% DSS vs controls. Loss of total sialylation was associated with reduced mucus thickness on fecal sections and within colon tissues. TM-IEC Slc35a1–/– mice showed altered microbiota with an increase in Clostridia disporicum, which is associated a global reduction in the abundance of at least 20 unique taxa; however, metabolomic analysis did not show any significant differences in short-chain fatty acid levels. Treatment with 5-fluorouracil (5-FU) led to more severe small intestine mucositis in the IEC Slc35a1–/– mice vs. WT littermates, which was associated with reduced Lgr5+ cell representation in small intestinal crypts in IEC Slc35a1-/-;Lgr5-GFP mice.
Loss of overall sialylation impairs mucus stability and the stem cell niche leading to microbiota-dependent spontaneous colitis and tumorigenesis.
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