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HepG2 GalNAc-T1/2 Bump-and-hole secretome glycopeptide data

ABSTRACT: Engineered GalNAc-T glycosyltransferases were used to incorporate a chemically modified GalNAC analog into the secretome of HepG2 cells. The chemical modification included a bioorthogonal alkyne tag that allowed for introduction of a clickable, acid-cleavable biotin-picolyl-azide. Glycoproteins were enriched using Streptavidin, and on-bead digestion yielded solid phase-bound glycopeptides that were released with acid. Glycopeptides were analysed.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte, Cell Culture

SUBMITTER: Benjamin Schumann

LAB HEAD: Benjamin Schumann

PROVIDER: PXD018048 | Pride | 2020-04-22

REPOSITORIES: Pride

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			Action	DRS
	DataS1.xlsx	Xlsx
	DataS2.xlsx	Xlsx
	S1_BH-T1_DMSO.raw	Raw
	S1_BH-T1_cmpd_5.raw	Raw
	S1_BH-T2_DMSO.raw	Raw

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Publications

Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells.

Schumann Benjamin B Malaker Stacy Alyse SA Wisnovsky Simon Peter SP Debets Marjoke Froukje MF Agbay Anthony John AJ Fernandez Daniel D Wagner Lauren Jan Sarbo LJS Lin Liang L Li Zhen Z Choi Junwon J Fox Douglas Michael DM Peh Jessie J Gray Melissa Anne MA Pedram Kayvon K Kohler Jennifer Jean JJ Mrksich Milan M Bertozzi Carolyn Ruth CR

Molecular cell 20200422 5

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context ...[more]

PMID: 32325029

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HepG2 secretome tagged with the sugar analogs GalNAz and GalNAzMe

Project description:Chemically modified analogs of GalNAc, termed GalNAz or GalNAzMe, were incorporated in secreted glycoproteins. The compounds contain bioorthogonal, clickable functional groups that allowed for enrichment using biotin. Chemical glycoproteomics was performed after on-bead digestion and cleavage of glycopeptides from the solid support.

2020-09-14 | PXD020648 | Pride

K-562 GalNAc-T1/2 Bump-and-hole Whole cell lysate proteomics data

Project description:Engineered GalNAc-T glycosyltransferases were used to incorporate a chemically modified GalNAc analog into the glycoproteins on the cell surface of K-562 cells. The chemical modification included a bioorthogonal alkyne tag that allowed for introduction of a clickable, acid-cleavable biotin-picolyl-azide. Glycoproteins were enriched using Streptavidin, and on-bead digestion yielded a peptide fraction that was analysed by mass spectrometry.

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Next generation unnatural monosaccharides reveal that ESRRBO GlcNAcylation enhances pluripotency of mouse embryonic stem cells

Project description:Unnatural monosaccharides such as azidosugars that can be metabolically incorporated into cellular glycans are currently used as a major tool for glycan imaging and glycoproteomic profiling. As a common practice to enhance membrane permeability and cellular uptake, the unnatural sugars are per O acetylated, which, however, can induce a long-overlooked side reaction, non enzymatic S-glycosylation. Herein, we develop1,3 di esterified N azidoacetylgalactosamine (GalNAz) as the next generation chemical reporters for metabolic glycan labeling. Both 1,3 di O-acetylated GalNAz (1,3-Ac2GalNAz) and1,3 di O propionylated GalNAz (1,3-Pr2GalNAz)exhibited high efficiency for labeling protein O-GlcNAcylation with no artificial S-glycosylation. Applying1,3 Pr2GalNAz in mouse embryonic stem cells (mESCs), we identified ESRRB, a critical transcription factor for pluripotency, as an O-GlcNAcylated protein. We showed that ESRRB O-GlcNAcylation is important for mESC self-renewal and pluripotency. Mechanistically, ESRRB is O-GlcNAcylated by O-GlcNAc transferase (OGT) at serine 25 (Ser 25), which stabilizes ESRRB, promotes its transcription activity and facilitates its interactions with two master pluripotency regulators, OCT4 and NANOG.

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Next generation unnatural monosaccharides reveal that ESRRBO GlcNAcylation enhances pluripotency of mouse embryonic stem cells

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Cell-type specific tagging of glycoproteins in murine-human co-culture samples glycoproteomics lysates

Project description:We report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway (mut-AGX1/NahK/Nh-GalNAc-T2) that transforms an alkyne tagged sugar into the corresponding nucleotide-sugar. Only transfected cells incorporate the bioorthogonal sugar into glycoproteins in the presence of non-transfected cells. The incorporation of alkyne-sugar in glycoproteins allows the installation, by copper(I)-catalyzed reaction, of an acid cleavable biotinylated tag. Glycoproteins are enriched using Streptavidin and. after on-bead digestion, glycopeptides are eluted in acid-conditions to be then analysed by mass spectrometry.

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On-tissue spatially-resolved glycoproteomics guided by N-glycan imaging reveal global dysregulation of canine glioma glycoproteomic landscape

Project description:Here we present an approach to identify N-linked glycoproteins and deduce their spatial localization using a combination of MALDI N-glycan MSI and spatially-resolved glycoproteomics. We subjected glioma biopsies to on-tissue PNGaseF digestion and MALDI-MSI and found that the glycan HexNAc4-Hex5-NeuAc2 was predominantly expressed in necrotic regions of high-grade canine gliomas. To determine the underlying sialo-glycoprotein, various regions in adjacent tissue sections were subjected to microdigestion and manual glycoproteomic analysis. Results identified haptoglobin as the protein associated with HexNAc4-Hex5-NeuAc2, making our study the first report that directly links glycan imaging with intact glycopeptide identification. In total, our spatially-resolved glycoproteomics technique identified over 400 N-, O-, and S- glycopeptides from over 30 proteins, demonstrating the diverse array of glycosylation present on the tissue slides and the sensitivity of our technique. Ultimately, this proof-of-principle work demonstrates that spatially-resolved glycoproteomics greatly complement MALDI-MSI in understanding dysregulated glycosylation.

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Design of a mucin-selective protease for targeted degradation of cancer-associated mucins

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2023-07-21 | PXD042243 | Pride

Mutational screens highlight glycosylation as a modulator of colony-stimulating factor 3 receptor (CSF3R) activity

Project description:The colony-stimulating factor 3 receptor (CSF3R) controls the growth of neutrophils, the most abundant type of white blood cell. In healthy neutrophils, signaling is dependent on CSF3R binding to its ligand, CSF3. A single amino acid mutation in CSF3R, T618I, instead allows for constitutive, ligand-independent cell growth and leads to a rare type of cancer called chronic neutrophilic leukemia (CNL). However, the disease mechanism is not well understood. Here, we investigated why this threonine to isoleucine substitution is the predominant mutation in CNL and how it leads to uncontrolled neutrophil growth. Using protein domain mapping, we demonstrated that the single CSF3R domain containing residue 618 is sufficient for ligand-independent activity. We then applied an unbiased mutational screening strategy focused on this domain and found that activating mutations are enriched at sites normally occupied by asparagine, threonine, and serine residues – the three amino acids which are commonly glycosylated. We confirmed glycosylation at multiple CSF3R residues by mass spectrometry, including the presence of GalNAc and Gal-GalNAc glycans at wild-type threonine 618. Using the same approach applied to other cell surface receptors, we identified an activating mutation, S489F, in the interleukin-31 receptor alpha chain (IL-31Rα). Combined, these results suggest a role for glycosylated hotspot residues in regulating receptor signaling, mutation of which can lead to ligand-independent, uncontrolled activity and human disease.

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