Identification of global inhibitors of cellular glycosylation
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture
SUBMITTER: 
Adnan Halim
LAB HEAD: Henrik Clausen
PROVIDER: PXD039334 | Pride | 2023-05-05
REPOSITORIES: Pride
Publications
Identification of global inhibitors of cellular glycosylation.
Sørensen Daniel Madriz DM Büll Christian C Madsen Thomas D TD Lira-Navarrete Erandi E Clausen Thomas Mandel TM Clark Alex E AE Garretson Aaron F AF Karlsson Richard R Pijnenborg Johan F A JFA Yin Xin X Miller Rebecca L RL Chanda Sumit K SK Boltje Thomas J TJ Schjoldager Katrine T KT Vakhrushev Sergey Y SY Halim Adnan A Esko Jeffrey D JD Carlin Aaron F AF Hurtado-Guerrero Ramon R Weigert Roberto R Clausen Henrik H Narimatsu Yoshiki Y
Nature communications 20230220 1
Small molecule inhibitors of glycosylation enzymes are valuable tools for dissecting glycan functions and potential drug candidates. Screening for inhibitors of glycosyltransferases are mainly performed by in vitro enzyme assays with difficulties moving candidates to cells and animals. Here, we circumvent this by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins. We focused on GalNAc-type O-glycosylation and selected the GalNAc-T11 isoe ...[more]
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Project description:Small molecule inhibitors of glycosylation enzymes can be valuable tools for dissecting glycan functions and present great potential as drug discovery candidates. Most screening campaigns for inhibitors of glycosyltransferases have been limited to in vitro enzyme assays using recombinant enzymes, which often have led to promising candidates that have been difficult to study further in cells and animals due to issues with permeability and toxicity. Here, we aimed to circumvent these issues by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins, which allowed us to narrow in on specific steps in individual glycosylation pathways. We focused on GalNAc-type O-glycosylation, and initially selected the GalNAc-T11 isoenzyme that selectively initiates O-glycosylation of the endocytic low density lipoprotein receptor (LDLR)-related proteins as target. In our screen with a limited small molecule compound library, we did not identify selective inhibitors of GalNAc-T11 or the family of GalNAc-T isoenzymes, however we identified two potent compounds that broadly inhibited glycosylation processes in a reversible manner. We demonstrate that these compounds inhibit all Golgi-localized glycosylation and suggest that this is mediated by reversible fragmentation of the Golgi system, presumably via interactions with HSP90. Finally, we demonstrate how these compounds can be used in cell model studies to dissect roles of different types of glycosylation by showing the importance of Tn-expression for binding of cancer-specific antibodies and by validating further the importance of cellular heparan sulfate in the binding and infection of SARS-CoV-2
2023-03-11 | PXD029623 | Pride
Project description:Most proteins trafficking the secretory pathway of metazoan cells will acquire GalNAc-type O-glycosylation. GalNAc-type O-glycosylation is differentially regulated in cells by the expression of a repertoire of up to twenty genes encoding polypeptide GalNAc-transferase isoforms (GalNAc-Ts) that initiate O-glycosylation by catalyzing the attachment of GalNAc residues to select Ser and Thr residues. These GalNAc-Ts orchestrate the positions and patterns of O-glycans on proteins in poorly understood coordinated ways guided partly by the kinetic properties and substrate specificities of their catalytic domains and modulatory effects of their unique GalNAc-binding lectin domains. Here, we provide the hereto most comprehensive characterization of the non-redundant contributions of individual GalNAc-T isoforms to the O-glycoproteome of the human HEK293 cell line using quantitative differential O-glycoproteomics on a panel of isogenic HEK293 cell lines with knockout of GalNAc-T genes (GALNT1, T2, T3, T7, T10, or T11). We confirm that a major part of the O-glycoproteome is covered by redundancy, while distinct subsets of O-glycosites are covered by non-redundant GalNAc-T isoform-specific functions. We demonstrate that GalNAc-T7 and T10 as predicted from in vitro studies function in completion of high density O-glycosylated regions, while GalNAc-T11 selectively controls the site-specific O-glycosylation of low-density lipoprotein-related receptors in the linker regions between the ligand-binding LDLR class A repeats.
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Project description:The GalNAc-type O-glycoproteome is orchestrated by a large family of polypeptide GalNAc-transferase isoenzymes (GalNAc-Ts) with partially overlapping contributions to the O-glycoproteome as well as distinct non-redundant functions. Increasing evidence indicate that individual GalNAc-Ts co-regulate and fine-tune specific protein functions in health and disease, and deficiencies in individual GALNT genes underlie congenital diseases with distinct phenotypes. Studies of GalNAc-T specificities have mainly been performed with in vitro enzyme assays using short peptide substrate, but recently quantitative differential O-glycoproteomics of isogenic cells with and without GALNT genes has enabled a more unbiased exploration of the non-redundant contributions of individual GalNAc-Ts. Both approaches suggest that fairly small subsets of O-glycosites are non-redundantly regulated by specific GalNAc-Ts, but how these isoenzymes orchestrate regulation amongst competing redundant substrates is unclear. To explore this, we developed isogenic cell model systems with Tet-On inducible expression of two GalNAc-T genes, GALNT2 and GALNT11, in a knockout background. Using quantitative O-glycoproteomics with TMT labelling we found that isoform-specific glycosites were glycosylated in a dose dependent manner, and induction of GalNAc-T2 or T11 produced discrete glycosylation effects without affecting the major part of the glycoproteome. The results support the findings that individual GalNAc-T isoenzymes can serve in fine-tuned regulation of distinct protein functions
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Project description:The Golgi stress response is a homeostatic mechanism that augments the functional capacity of the Golgi apparatus when Golgi function becomes insufficient (Golgi stress). Three response pathways of the Golgi stress response have been identified in mammalian cells, the TFE3, HSP47 and CREB3 pathways, which augment the capacity of specific Golgi functions such as N-glycosylation, anti-apoptotic activity and pro-apoptotic activity, respectively. On the contrary, glycosylation of proteoglycans (PGs) is another important function of the Golgi, although the response pathway upregulating expression of glycosylation enzymes for PGs in response to Golgi stress remains unknown. Here, we found that expression of glycosylation enzymes for PGs was induced upon insufficiency of PG glycosylation capacity in the Golgi (PG-Golgi stress), and that transcriptional induction of genes encoding glycosylation enzymes for PGs was independent of the known Golgi stress response pathways and ER stress response. Promoter analyses of genes encoding these glycosylation enzymes revealed the novel enhancer element PGSE, which regulates their transcriptional induction upon PG-Golgi stress. From these observations, the response pathway we discovered is a novel Golgi stress response pathway, which we have named the PG pathway.
2018-08-01 | GSE117938 | GEO
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2018-09-14 | GSE119909 | GEO
Project description:GalNAc-transferase (GalNAc-T) isoforms modify distinct subsets of the O-glycoproteome and GalNAc-type O-glycosylation is found on most proteins trafficking through the secretory pathway in metazoan cells. The O-glycoproteome is regulated by up to 20 polypeptide GalNAc-Ts and the contributions and biological functions of individual GalNAc-Ts are poorly understood. Here, we used a zinc-finger nuclease (ZFN)-directed knockout strategy to probe the contributions of the major GalNAc-Ts (GalNAc-T1 and T2) in liver cells, and explore how the GalNAc-T repertoire quantitatively affects the O-glycoproteome. We demonstrate that the majority of the O-glycoproteome is covered by redundancy, whereas distinct subsets of substrates are modified by non-redundant functions of GalNAc-T1 and T2. Differential transcriptomic analysis indicates that loss of function of a GalNAc-T induces specific transcriptional response. The non-redundant O-glycoproteome subsets for and the transcriptional responses for each isoform appeared to be related to different cellular processes, and for the GalNAc-T2 isoform supporting a role in lipid metabolism. The results demonstrate that GalNAc-Ts have non-redundant glycosylation functions, and that these may affect distinct cellular processes. The data provides a comprehensive resource for unique substrates for individual GalNAc-Ts. Our study provides a new view on the regulation of the O-glycoproteome, suggesting that the plurality of GalNAc-Ts arose to regulate distinct protein functions and cellular processes.
2015-09-30 | E-MTAB-3844 | biostudies-arrayexpress
Project description:Our studies provide direct evidence that O-glycosylation pathways play a role in the regulation of cell growth through apoptosis and proliferation pathways. Eight small molecular weight analogues of the GalNAc-alpha-1-O-serine/threonine structure based on 1-benzyl-2-acetamido-2- deoxy-alpha-O-D-galactopyranoside have been synthesised and tested in 5 human colorectal cancer cell lines. Three inhibitors, 1-benzyl-2-acetamido-2-deoxy-alpha-O-D-galactopyranoside and the corresponding 2-azido- and C-glycoside analogues, were screened in two colorectal cancer cell lines at 0.5mM and showed induction of apoptosis. Proliferation was down regulated in the same two cell lines with all three inhibitors, as detected by Ki67 staining and gene array. Treatment both cell lines with inhibitors led to changes in glycosylation detected with peanut lectin. The competitive action of the inhibitors resulted in the intracellular formation of 28 aryl-glycan products which were identified by MALDI and electrospray mass spectroscopy. The structures found map onto known O-glycosylation biosynthetic pathways and showed a differential pattern for each of the inhibitors in both cell lines. Gene array analysis of the glycogenes illustrated a pattern of glycosytransferases that matched the glycan structures found in glycoproteins and aryl-glycans formed in the PC/AA/C1/SB10C cells, however there was no action of the three inhibitors on glycogene transcript levels. The inhibitors act at both intermediary metabolic and genomic levels, resulting in altered protein glycosylation and arylglycan formation. These events may play a part in growth arrest. Keywords: Response to inhibitors, Apoptosis, Aryl-glycans, Benzyl-O-GalNAc, Growth Inhibition, O-Glycans
2008-08-18 | GSE12422 | GEO
Project description:Our studies provide direct evidence that O-glycosylation pathways play a role in the regulation of cell growth through apoptosis and proliferation pathways. Eight small molecular weight analogues of the GalNAc-alpha-1-O-serine/threonine structure based on 1-benzyl-2-acetamido-2- deoxy-alpha-O-D-galactopyranoside have been synthesised and tested in 5 human colorectal cancer cell lines. Three inhibitors, 1-benzyl-2-acetamido-2-deoxy-alpha-O-D-galactopyranoside and the corresponding 2-azido- and C-glycoside analogues, were screened in two colorectal cancer cell lines at 0.5mM and showed induction of apoptosis. Proliferation was down regulated in the same two cell lines with all three inhibitors, as detected by Ki67 staining and gene array. Treatment both cell lines with inhibitors led to changes in glycosylation detected with peanut lectin. The competitive action of the inhibitors resulted in the intracellular formation of 28 aryl-glycan products which were identified by MALDI and electrospray mass spectroscopy. The structures found map onto known O-glycosylation biosynthetic pathways and showed a differential pattern for each of the inhibitors in both cell lines. Gene array analysis of the glycogenes illustrated a pattern of glycosytransferases that matched the glycan structures found in glycoproteins and aryl-glycans formed in the PC/AA/C1/SB10C cells, however there was no action of the three inhibitors on glycogene transcript levels. The inhibitors act at both intermediary metabolic and genomic levels, resulting in altered protein glycosylation and arylglycan formation. These events may play a part in growth arrest. Experiment Overall Design: The effects of the O-glycan inhibitors Benzyl 2-azido-2-deoxy-alpha-D-galactopyranoside (alpha-OBn GalN3), Benzyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside (alpha-OBn GalNAc), and 2-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-1-phenylethane (alpha-CBn GalNAc) on the colorectal cancer cell line PC/AA/C1/SB10C were examined. Experiment Overall Design: The PC/AA/C1/SB10C cells were treated with with 0.5 mM of the inhibitors for four days. In control experiments cells were cultivated without inhibitor. All experiments were performed twice. Total RNA was extracted with RNAzol reagent in accordance to the manufacturer instructions (PeQLab, Biotechnology Fareham, UK), treated with RNase-free DNase (DNA-free, Ambion,Warrington, UK) and integrity verified using the Agilent Bioanalyzer (Agilent Technologies, West Lothian, UK), by the presence of the 28S and 18S rRNA on agarose gels and an A260/280 ratio in the range of 1.9â2.1. A sample (5 μg) of total RNA was used for production of biotinylated cRNA as described in the Affymetrix GeneChip analysis instruction manual (Affymetrix UK, High Wycombe, UK). The human genome 133A 2.0 array was then hybridized with the biotin-labeled cRNA fragments for 16 h at 45°C. Washing steps for the chip, staining with streptavidin-phycoerythrin, signal amplification and scanning were performed according to the manufacturerâs instructions (Affymetrix UK, High Wycombe, UK). Signal values were exported with the GeneChip operating software (GCOS, Affymetrix). Further analyses were performed with the software âCorrXpressionâ, which is described in detail elsewhere (Klein et al. 2005, J Mol Med. 83:362-376; Wessel et al. 2006, In Silico Biol.6:61-70).
2008-10-25 | E-GEOD-12422 | biostudies-arrayexpress
Project description:Dr. Clausen's laboratory is interested in the structure, biosynthesis and genetic regulation of glycoconjugates, with a major focus on mucins. This laboratory is studying the glycosylation and secretion process of mucins and biological functions involving mucins. This lab is also interested in receptor modulation mediated by glycosylation or through glycosphingolipids. RNA samples from two human pancreatic cell lines. The relevance of the variable expression levels of isoforms belonging to the polypeptide GalNAc-transferase gene-family is still a puzzle to us. The polypeptide GalNAc-transferase gene family is estimated to contain 24 gene-members all participating in the initiation of mucin-type O-glycosylation. It has become apparent that each of these individual isoforms have different tissue distribution profiles and kinetic capabilities. Mucin-type O-glycosylation is thus controlled, and dependent upon the repetoir of expressed GalNAc-transferases in any given organ and cell type at any given time. We have been trying to contribute to the understanding of the ordered process controlling mucin-type O-glycosylation in both normal and malignant models, and are currently investigating a pancreatic cell line model in the lab. The pancreatic tumor cell line ASPC-1 has been found to possess low metastatic potential in mouse models, in contrast to the pancreatic tumor cell line Suit-T2 possessing high metastatic potential. Immunohistochemical analysis of both cell lines, ASPC-1 and Suit-T2, using our monoclonal antibodies to 7 isoforms has revealed great differences in the expression profile of these 7 isoforms in theses two cell lines, and for instance GalNAc-T3 has been found to be highly expressed in Suit-T2 but completely lacking in ASPC-1. Two cell lines: ASPC-1 and Suit-T2 are analyzed with 3 independent replicate prepared for each cell line.
2010-12-11 | E-GEOD-26011 | biostudies-arrayexpress
Project description:Dr. Clausen's laboratory is interested in the structure, biosynthesis and genetic regulation of glycoconjugates, with a major focus on mucins. This laboratory is studying the glycosylation and secretion process of mucins and biological functions involving mucins. This lab is also interested in receptor modulation mediated by glycosylation or through glycosphingolipids. RNA samples from two human pancreatic cell lines. The relevance of the variable expression levels of isoforms belonging to the polypeptide GalNAc-transferase gene-family is still a puzzle to us. The polypeptide GalNAc-transferase gene family is estimated to contain 24 gene-members all participating in the initiation of mucin-type O-glycosylation. It has become apparent that each of these individual isoforms have different tissue distribution profiles and kinetic capabilities. Mucin-type O-glycosylation is thus controlled, and dependent upon the repetoir of expressed GalNAc-transferases in any given organ and cell type at any given time. We have been trying to contribute to the understanding of the ordered process controlling mucin-type O-glycosylation in both normal and malignant models, and are currently investigating a pancreatic cell line model in the lab. The pancreatic tumor cell line ASPC-1 has been found to possess low metastatic potential in mouse models, in contrast to the pancreatic tumor cell line Suit-T2 possessing high metastatic potential. Immunohistochemical analysis of both cell lines, ASPC-1 and Suit-T2, using our monoclonal antibodies to 7 isoforms has revealed great differences in the expression profile of these 7 isoforms in theses two cell lines, and for instance GalNAc-T3 has been found to be highly expressed in Suit-T2 but completely lacking in ASPC-1.
2010-12-11 | GSE26011 | GEO