TAILS N-terminomics and proteomics reveal complex regulation of proteolytic cleavage by O-glycosylation

King, Sarah L.; Goth, Christoffer K.; Eckhard, Ulrich; Joshi, Hiren J.; Haue, Amalie Dahl; Vakhrushev, Sergey Y.; Schjoldager, Katrine T.-B. G.; Overall, Christopher M.; Wandall, Hans H.

doi:10.1074/jbc.ra118.001978

Cited by 29 publications

(32 citation statements)

References 80 publications

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“…We previously developed techniques that allow the identification of site‐specific O‐glycosylation on a proteome‐wide basis, which has enabled high‐throughput mapping of sites of O‐glycan attachment in proteomes from both cell lines and tissues . Furthermore, we have shown that targeted KO of individual GALNT s allows for discovery of GalNAc‐T isoform‐specific substrates . Two approaches were used to characterize the differential O‐glycoproteomes of ΔT1, ΔT2, and ΔT3.…”

Section: Resultsmentioning

confidence: 99%

O‐glycan initiation directs distinct biological pathways and controls epithelial differentiation

Bagdonaite

Pallesen

et al. 2020

EMBO Reports

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Post‐translational modifications (PTMs) greatly expand the function and potential for regulation of protein activity, and O‐glycosylation is among the most abundant and diverse PTMs. Initiation of O‐GalNAc glycosylation is regulated by 20 distinct GalNAc‐transferases (GalNAc‐Ts), and deficiencies in individual GalNAc‐Ts are associated with human disease, causing subtle but distinct phenotypes in model organisms. Here, we generate a set of isogenic keratinocyte cell lines lacking either of the three dominant and differentially expressed GalNAc‐Ts. Through the ability of keratinocytes to form epithelia, we investigate the phenotypic consequences of the loss of individual GalNAc‐Ts. Moreover, we probe the cellular responses through global transcriptomic, differential glycoproteomic, and differential phosphoproteomic analyses. We demonstrate that loss of individual GalNAc‐T isoforms causes distinct epithelial phenotypes through their effect on specific biological pathways; GalNAc‐T1 targets are associated with components of the endomembrane system, GalNAc‐T2 targets with cell–ECM adhesion, and GalNAc‐T3 targets with epithelial differentiation. Thus, GalNAc‐T isoforms serve specific roles during human epithelial tissue formation.

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Section: Resultsmentioning

confidence: 99%

O‐glycan initiation directs distinct biological pathways and controls epithelial differentiation

Bagdonaite

Pallesen

et al. 2020

EMBO Reports

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“…Over the past 10 years numerous studies have highlighted how various PTMs impact eukaryote biology. In eukaryotes, protease activity plays a critical role in homeostasis and health by maintaining strict control over protein and cellular function [37][38][39][40] . In prokaryote biology, however, systems analysis of proteolysis is in its infancy 41 .…”

Section: Discussionmentioning

confidence: 99%

Protein cleavage influences surface protein presentation in Mycoplasma pneumoniae

Berry

Widjaja

Jarocki

et al. 2021

Sci Rep

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Mycoplasma pneumoniae is a significant cause of pneumonia and post infection sequelae affecting organ sites distant to the respiratory tract are common. It is also a model organism where extensive ‘omics’ studies have been conducted to gain insight into how minimal genome self-replicating organisms function. An N-terminome study undertaken here identified 4898 unique N-terminal peptides that mapped to 391 (56%) predicted M. pneumoniae proteins. True N-terminal sequences beginning with the initiating methionine (iMet) residue from the predicted Open Reading Frame (ORF) were identified for 163 proteins. Notably, almost half (317; 46%) of the ORFS derived from M. pneumoniae strain M129 are post-translationally modified, presumably by proteolytic processing, because dimethyl labelled neo-N-termini were characterised that mapped beyond the predicted N-terminus. An analysis of the N-terminome describes endoproteolytic processing events predominately targeting tryptic-like sites, though cleavages at negatively charged residues in P1′ (D and E) with lysine or serine/alanine in P2′ and P3′ positions also occurred frequently. Surfaceome studies identified 160 proteins (23% of the proteome) to be exposed on the extracellular surface of M. pneumoniae. The two orthogonal methodologies used to characterise the surfaceome each identified the same 116 proteins, a 72% (116/160) overlap. Apart from lipoproteins, transporters, and adhesins, 93/160 (58%) of the surface proteins lack signal peptides and have well characterised, canonical functions in the cell. Of the 160 surface proteins identified, 134 were also targets of endo-proteolytic processing. These processing events are likely to have profound implications for how the host immune system recognises and responds to M. pneumoniae.

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“…In addition to the above-mentioned glycoproteomics studies performed on glycoprotein samples from unaltered biological sources, the SimpleCell technology, which manipulates cells to homogenously express truncated O -GalNAc glycans (Tn and sialyl-Tn (sTn)) on mucins and mucin-like domains due to the inactivation of the chaperone COSMC , represents an important innovation relevant for glycoproteomics [ 84–88 ]. The SimpleCell technology has not only opened exciting avenues to study the challenging mucin glycobiology including how O -glycans impact proteolytic cleavage events, the substrate-specificity of GalNAc-transferases and the role of O -GalNAc glycans in cell–cell interactions and cell differentiation [ 89–93 ], but has also improved our ability to profile the critically underexplored O -glycoproteome. Glycoproteomics efforts using the SimpleCell technology to study mucin-type glycosylation in different human and other mammalian cell lines have led to an impressive database ( http://www.glycoproteomics.somee.com/ ), comprising a total of 18 397 unique O -glycopeptides spanning 10 197 O -glycan sites from 2039 O -glycoproteins within the human O -glycoproteome supported by glycopeptide MS/MS evidence.…”

Section: Recent N - and O -Glycmentioning

confidence: 99%

Towards structure-focused glycoproteomics

Chernykh

Kawahara

Thaysen‐Andersen

2021

Biochemical Society Transactions

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Facilitated by advances in the separation sciences, mass spectrometry and informatics, glycoproteomics, the analysis of intact glycopeptides at scale, has recently matured enabling new insights into the complex glycoproteome. While diverse quantitative glycoproteomics strategies capable of mapping monosaccharide compositions of N- and O-linked glycans to discrete sites of proteins within complex biological mixtures with considerable sensitivity, quantitative accuracy and coverage have become available, developments supporting the advancement of structure-focused glycoproteomics, a recognised frontier in the field, have emerged. Technologies capable of providing site-specific information of the glycan fine structures in a glycoproteome-wide context are indeed necessary to address many pending questions in glycobiology. In this review, we firstly survey the latest glycoproteomics studies published in 2018–2020, their approaches and their findings, and then summarise important technological innovations in structure-focused glycoproteomics. Our review illustrates that while the O-glycoproteome remains comparably under-explored despite the emergence of new O-glycan-selective mucinases and other innovative tools aiding O-glycoproteome profiling, quantitative glycoproteomics is increasingly used to profile the N-glycoproteome to tackle diverse biological questions. Excitingly, new strategies compatible with structure-focused glycoproteomics including novel chemoenzymatic labelling, enrichment, separation, and mass spectrometry-based detection methods are rapidly emerging revealing glycan fine structural details including bisecting GlcNAcylation, core and antenna fucosylation, and sialyl-linkage information with protein site resolution. Glycoproteomics has clearly become a mainstay within the glycosciences that continues to reach a broader community. It transpires that structure-focused glycoproteomics holds a considerable potential to aid our understanding of systems glycobiology and unlock secrets of the glycoproteome in the immediate future.

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TAILS N-terminomics and proteomics reveal complex regulation of proteolytic cleavage by O-glycosylation

Abstract: ABSTRACT

Cited by 29 publications

References 80 publications

O‐glycan initiation directs distinct biological pathways and controls epithelial differentiation

O‐glycan initiation directs distinct biological pathways and controls epithelial differentiation

Protein cleavage influences surface protein presentation in Mycoplasma pneumoniae

Towards structure-focused glycoproteomics

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