Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology

Steentoft, Catharina; Vakhrushev, Sergey Y.; Joshi, Hiren J.; Kong, Yun; Vester-Christensen, Malene Bech; Schjoldager, Katrine T-Bg; Lavrsen, Kirstine; Dabelsteen, Sally; Pedersen, Nis Borbye; Marcos-Silva, Lara; Gupta, Ramneek; Bennett, Eric; Mandel, Ulla; Brunak, Søren; Wandall, Hans H.; Levery, Steven B.; Clausen, Henrik

doi:10.1038/emboj.2013.79

Cited by 1,246 publications

(1,187 citation statements)

References 50 publications

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“…We used the human MDA-MB-231 breast cancer cell line because we previously identified O-GalNAc glycosites in the mucin-linker domain of α-DG in MDA-MB-231 O-GalNAc SimpleCells (21). α-DG contains both O-Man and O-GalNAc glycans in this region (22)(23)(24), and some sites are potentially occupied by either type of glycosylation (25).…”

Section: Resultsmentioning

confidence: 99%

“…"Bottomup" higher-energy collision-induced dissociation-electron-transfer dissociation (HCD/ETD)-based mass spectrometric analysis has allowed us to provide a first draft of the human O-GalNAc glycoproteome where we expanded the knowledge of O-glycoproteins and O-glycosites many fold (21). Here, we designed a similar strategy to simplify O-Man glycans in a human breast cancer cell line, which allowed us to probe the O-Man glycoproteome.…”

Section: Significancementioning

confidence: 99%

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Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins

Vester-Christensen¹,

Halim²,

Joshi³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The metazoan O-mannose (O-Man) glycoproteome is largely unknown. It has been shown that up to 30% of brain O-glycans are of the O-Man type, but essentially only alpha-dystroglycan (α-DG) of the dystrophin-glycoprotein complex is well characterized as an O-Man glycoprotein. Defects in O-Man glycosylation underlie congenital muscular dystrophies and considerable efforts have been devoted to explore this O-glycoproteome without much success. Here, we used our SimpleCell strategy using nuclease-mediated gene editing of a human cell line (MDA-MB-231) to reduce the structural heterogeneity of O-Man glycans and to probe the OMan glycoproteome. In this breast cancer cell line we found that O-Man glycosylation is primarily found on cadherins and plexins on β-strands in extracellular cadherin and Ig-like, plexin and transcription factor domains. The positions and evolutionary conservation of O-Man glycans in cadherins suggest that they play important functional roles for this large group of cell adhesion glycoproteins, which can now be addressed. The developed O-Man SimpleCell strategy is applicable to most types of cell lines and enables proteome-wide discovery of O-Man protein glycosylation.POMGnT1 | O-glycosylation | Orbitrap | mass spectrometry | glycoproteomics

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Section: Resultsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins

Vester-Christensen¹,

Halim²,

Joshi³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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148

187

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“…Based on prediction software (34) and the best known O-GlcNAcylation motif (22), O-GlcNAcylation is predicted to occur on Ser202, Thr205, and Ser208 in Tau ND, no O-GlcNAcylation detected; x, two heptad repeats from RNA polymerase II; thus this 14mer peptide represents any/all of these repeats. *Proteins with (S/T)PX(S/T) motif for which evidence exists that the first S/T can be phosphorylated.…”

Section: Reciprocal Interplay Does Not Occur On Ser/thr Sites Targetementioning

confidence: 99%

Elucidating crosstalk mechanisms between phosphorylation and O-GlcNAcylation

Leney

Atmioui

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

138

127

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Proteins can be modified by multiple posttranslational modifications (PTMs), creating a PTM code that controls the function of proteins in space and time. Unraveling this complex PTM code is one of the great challenges in molecular biology. Here, using mass spectrometry-based assays, we focus on the most common PTMs-phosphorylation and O-GlcNAcylation-and investigate how they affect each other. We demonstrate two generic crosstalk mechanisms. First, we define a frequently occurring, very specific and stringent phosphorylation/ O-GlcNAcylation interplay motif, (pSp/T)P(V/A/T)(gS/gT), whereby phosphorylation strongly inhibits O-GlcNAcylation. Strikingly, this stringent motif is substantially enriched in the human (phospho)proteome, allowing us to predict hundreds of putative O-GlcNAc transferase (OGT) substrates. A set of these we investigate further and show them to be decent substrates of OGT, exhibiting a negative feedback loop when phosphorylated at the P-3 site. Second, we demonstrate that reciprocal crosstalk does not occur at PX(S/T)P sites, i.e., at sites phosphorylated by proline-directed kinases, which represent 40% of all sites in the vertebrate phosphoproteomes.O-GlcNAcylation | phosphorylation | crosstalk | signaling | regulation

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“…However, some potential O-glycosylation sites were detected with several O-glycosylation site prediction programs such as NetOGlyc 4.0 Server. 30) The specific activities of the native and recombinant EaChiA were 8.9 × 10 9 and 9.4 × 10 9 units/mol enzyme, respectively, using glycol chitin as the substrate. As shown in Fig.…”

Section: Preparation and Characterization Of Recombinant Eachiamentioning

confidence: 99%

Purification, cDNA cloning, and characterization of LysM-containing plant chitinase from horsetail (Equisetum arvense)

Inamine

Onaga

Ohnuma

et al. 2015

Bioscience, Biotechnology, and Biochemistry

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Chitinase-A (EaChiA), molecular mass 36 kDa, was purified from the vegetative stems of a horsetail (Equisetum arvense) using a series of column chromatography. The N-terminal amino acid sequence of EaChiA was similar to the lysin motif (LysM). A cDNA encoding EaChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1320 nucleotides and encoded an open reading frame of 361 amino acid residues. The deduced amino acid sequence indicated that EaChiA is composed of a N-terminal LysM domain and a C-terminal plant class IIIb chitinase catalytic domain, belonging to the glycoside hydrolase family 18, linked by proline-rich regions. EaChiA has strong chitin-binding activity, however, no antifungal activity. This is the first report of a chitinase from Equisetopsida, a class of fern plants, and the second report of a LysM-containing chitinase from a plant.

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Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology

Cited by 1,246 publications

References 50 publications

Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins

Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins

Elucidating crosstalk mechanisms between phosphorylation and O-GlcNAcylation

Purification, cDNA cloning, and characterization of LysM-containing plant chitinase from horsetail (Equisetum arvense)

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