Dataset Information

Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines

ABSTRACT: 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

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Sergey Vakhrushev

LAB HEAD: Sergey Vakhrushev

PROVIDER: PXD010155 | Pride | 2018-10-16

REPOSITORIES: Pride

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			Action	DRS
	171020_Fusion_LC2_John_Hek_WT_T11_inducer_Trypsin_TMT6plex_PNA_2h_ETD_HCD_High_pH.xlsx	Xlsx
	171020_Fusion_LC2_John_Hek_WT_T11_inducer_Trypsin_TMT6plex_PNA_2h_ETD_HCD_High_pH_1.raw	Raw
	171020_Fusion_LC2_John_Hek_WT_T11_inducer_Trypsin_TMT6plex_PNA_2h_ETD_HCD_High_pH_2.raw	Raw
	171020_Fusion_LC2_John_Hek_WT_T11_inducer_Trypsin_TMT6plex_PNA_2h_ETD_HCD_High_pH_3.raw	Raw
	171020_Fusion_LC2_John_Hek_WT_T11_inducer_Trypsin_TMT6plex_PNA_2h_ETD_HCD_High_pH_4.raw	Raw

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Publications

Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines.

Hintze John J Ye Zilu Z Narimatsu Yoshiki Y Madsen Thomas Daugbjerg TD Joshi Hiren J HJ Goth Christoffer K CK Linstedt Adam A Bachert Collin C Mandel Ulla U Bennett Eric P EP Vakhrushev Sergey Y SY Schjoldager Katrine T KT

The Journal of biological chemistry 20181016 49

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 besides distinct nonredundant functions. Increasing evidence indicates 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 s ...[more]

PMID: 30327431

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Project description:A large family of GalNAc transferases (GalNAc-Ts) catalyzes the covalent attachment of N-Acetylgalactosamine (GalNAc) to serine and threonine residues on proteins that pass through the secretory pathway in the first committed step of mucin-type O-glycosylation. Abnormalities in the activity of individual GalNAc-Ts can result in congenital disorders of O-glycosylation (CDG) and influence other biological functions. Although ~85% of proteins that pass through the secretory pathway are modified with O-glycans, only 47 O-glycosylation sites (O-glycosites) from 22 mouse glycoproteins and 17 publications are present on UniProt and an O-glycoprotein database (http://www.oglyp.org/). A compilation of all in vivo O-glycosites (the O-glycoproteome) would be an invaluable step in determining the function of proteins decorated with N-Acetylgalactosamine and what role(s), if any, the O-glycans play. While approaches to delineate O-glycosites have been developed for simple, genetically engineered cell lines, there is no universal approach to mapping the O-glycosites of glycoproteins in complex tissue extracts or biological fluids. We have developed chemical and enzymatic conditions that cleave solitary O-glycans while leaving the modified core protein/peptides assayable by mass spectrometry (MS). The methodology permits the mapping of thousands of O-glycosites decorated with Tn or T antigen from tissues or biological fluids. We then integrated an HCD-pd-EThcD MS workflow and software, including MSFragger-Glyco, pGlyco3, and O-Pair, to study the mouse brain, heart, lung, liver, spleen, kidney, colon, muscle, submandibular gland, and whole blood. The integrated approach identified 2154 solitary O-glycosites from 595 glycoproteins. The O-glycosites and glycoproteins displayed consensus motifs and Gene Ontology (GO) terms for O-glycoproteins. Limited overlap of O-glycosites was observed with protein O-GlcNAcylation and phosphorylation sites. Integrating quantitative glycoproteomics and proteomics revealed a tissue-specific regulation of O-glycosites that the differential expression of Galnt isoenzymes in tissues partly contributes to. We next used established quantitative glycoproteomics and proteomics methods to identify site-specific substrates that may contribute to biologically relevant phenotypes when Galnt2 was genetically ablated in a Galnt2-null mouse model, which presents with lipid and metabolic dysregulation. Our findings suggest networks of Galnt2 direct and indirect interactions that may explain the complex metabolic phenotypes. The mouse O-glycoproteome-map and quantitative glycoproteomics/proteomics approach will provide a valuable foundation for revealing the significance of O-glycosylation biology in CDGs and other diseases and conditions.

Project description:Glycosylation is an abundant post-translational modification of both intracellular and extracellular proteins [1]. The majority of glycans are classified as N-linked chains, where the carbohydrate moiety is attached to asparagine residues, or O-linked chains, most commonly linked to a serine or threonine. N-linked glycosylation is initiated by the oligosaccharyltransferase complex with only two paralogs of the catalytic subunit, whereas O-glycan initiation is more complex. There are several types of O-linked glycosylation, but among the most diverse is the mucin or GalNAc type (hereafter referred to as O-glycosylation). O-glycosylation is initiated by 20 evolutionarily conserved polypeptide GalNAc-transferases (GalNAc-Ts), which catalyze the first step in the O-glycosylation of proteins by adding GalNAc residues to threonine, serine, and tyrosine amino acids (Fig 1A). Each of the GalNAc-Ts are differentially expressed in various tissues and have both distinct and overlapping peptide substrate specificities [2-12]. Thus, the repertoire of GalNAc-Ts expressed in a given cell determines the subset and O-glycosite pattern of glycosylated proteins [13]. Substantial efforts have been made to characterize and predict the substrate specificities of GalNAc-Ts in vitro, but understanding of the in vivo specificities of the individual GalNAc-Ts or their biological functions is limited [13-15]. This lack of insight prevents an understanding of how site-specific O-linked glycosylation affects diseases, such as metabolic disorders, cardiovascular disease, and various malignancies, that have been associated with GalNAc-Ts through genome-wide association studies and other linkage studies [16-26]. Therefore, it is imperative that we establish how O-glycosylation at specific sites in proteins affects protein function. A major task in achieving this goal is to identify the non-redundant biological functions of site-specific O-glycosylation. We and others recently developed new strategies for identifying specific sites on proteins that undergo O-glycosylation in different cell types and tissues [27-31]. Characterization of the O-glycoproteomic landscape in isolated human cells and multiple human cell lines suggests that more than 80 % of all proteins that traffic through the secretory pathway are O-glycoproteins [28, 30]. Probing the non-redundant contributions of individual GalNAc-Ts in cells with and without specific GalNAc-Ts [32-34] has revealed broad substrate specificities for some of the individual isoforms, whereas others seem to have very restricted substrate specificities [33-35]. Assessing all of the mapped O-glycosylation sites to identify associations between O-glycosites and protein annotations, we recently found that O-glycans are over-represented close to tandem repeat regions, protease cleavage sites, within propeptides, and on a select group of protein domains [28, 30, 36]. Although such general associations between the location of O-glycans and protein functions may direct future investigations, the strategy does not define the function of site-specific glycosylation. Further progress in discovering and defining novel functions of site-specific glycosylation events requires direct quantitative analysis of potential biological responses induced by the loss of distinct GalNAc-T isoforms, and such biological responses are not easily observed in single cell culture systems. Instead, more complex model systems can be used to examine and dissect the molecular mechanisms underlying the important biological functions of site-specific glycosylation. We previously used an organotypic tissue model equipped with genetically engineered cells to decipher the function of elongated O-glycans [29]. In the present study, we use the model combined with quantitative O-glycoproteomics and phosphoproteomics to perform open-ended discovery of the biological functions of site-specific glycosylation governed by GalNAc-Ts (Fig 1B). With this combinatorial strategy, we demonstrate that loss of individual GalNAc-T isoforms has distinct phenotypic consequences through their effect on distinct biological pathways, suggesting specific roles during epithelial formation.

Dataset Information

Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines

Dataset's files

Publications

Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the <i>O</i>-glycoproteome by inducible expression in isogenic cell lines.

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