Dataset Information

Identification of global inhibitors of cellular glycosylation

ABSTRACT: 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 isoenzyme that selectively glycosylates the endocytic low density lipoprotein receptor (LDLR)-related proteins as target. Our screen of a limited small molecule compound library did not identify selective inhibitors of GalNAc-T11, however we identified two compounds that broadly inhibited Golgi-localized glycosylation processes. These compounds mediated reversible fragmentation of the Golgi system without affecting secretion. We demonstrate how these inhibitors can be used to manipulate glycosylation in cells to induce expression of truncated O-glycans and augment binding of cancer-specific Tn-glycoprotein antibodies and to inhibit expression of heparan sulfate and binding and infection of SARS-CoV-2.

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

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Dataset's files

Source:

			Action	DRS
	201012_Lumos__LC2100__DANIEL_ecad_DMSO_5_1_90min_ETD_HCD_20201013132242.raw	Raw
	201012_Lumos__LC2100__DANIEL_ecad_DMSO_5_1_90min_HCD2.msf	Msf

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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]

PMID: 36804936

Similar Datasets

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 Î¼g) 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 45Â°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).