Dataset Information

Deconstruction of the GalNAc-Type O-Glycosylation Capacity of Human HEK293 Cells

ABSTRACT: Most proteins trafficking the secretory pathway of metazoan cells will acquire GalNAc-type O-glycosylation. GalNAc-type O-glycosylation is differentially regulated in cells by the expression of a repertoire of up to twenty genes encoding polypeptide GalNAc-transferase isoforms (GalNAc-Ts) that initiate O-glycosylation by catalyzing the attachment of GalNAc residues to select Ser and Thr residues. These GalNAc-Ts orchestrate the positions and patterns of O-glycans on proteins in poorly understood coordinated ways guided partly by the kinetic properties and substrate specificities of their catalytic domains and modulatory effects of their unique GalNAc-binding lectin domains. Here, we provide the hereto most comprehensive characterization of the non-redundant contributions of individual GalNAc-T isoforms to the O-glycoproteome of the human HEK293 cell line using quantitative differential O-glycoproteomics on a panel of isogenic HEK293 cell lines with knockout of GalNAc-T genes (GALNT1, T2, T3, T7, T10, or T11). We confirm that a major part of the O-glycoproteome is covered by redundancy, while distinct subsets of O-glycosites are covered by non-redundant GalNAc-T isoform-specific functions. We demonstrate that GalNAc-T7 and T10 as predicted from in vitro studies function in completion of high density O-glycosylated regions, while GalNAc-T11 selectively controls the site-specific O-glycosylation of low-density lipoprotein-related receptors in the linker regions between the ligand-binding LDLR class A repeats.

INSTRUMENT(S): Orbitrap Fusion ETD, LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Sergey Vakhrushev

LAB HEAD: Sergey Y. Vakhrushev

PROVIDER: PXD009955 | Pride | 2019-06-24

REPOSITORIES: pride

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Source:

			Action	DRS
	131023_YN_Hek293xxxxxx_diff_T7KO_sec_VVA_2h_HCD_ETD_95_IEF1.raw	Raw
	131023_YN_Hek293xxxxxx_diff_T7KO_sec_VVA_2h_HCD_ETD_95_IEF10.raw	Raw
	131023_YN_Hek293xxxxxx_diff_T7KO_sec_VVA_2h_HCD_ETD_95_IEF11.raw	Raw
	131023_YN_Hek293xxxxxx_diff_T7KO_sec_VVA_2h_HCD_ETD_95_IEF12.raw	Raw
	131023_YN_Hek293xxxxxx_diff_T7KO_sec_VVA_2h_HCD_ETD_95_IEF1_12ETD_full_semi.msf	Msf

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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.

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Deconstruction of the GalNAc-Type O-Glycosylation Capacity of Human HEK293 Cells

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