Dataset Information

TAILS N-Terminomics and Proteomics Show Complex Regulation of Proteolytic Processing by O-glycosylation

ABSTRACT: Proteolytic processing is an irreversible post-translational modification functioning as a ubiquitous regulator of cellular activity. Protease activity is tightly regulated by control of gene expression, compartmentalisation of enzyme and substrate, zymogen activation, enzyme inactivation, and substrate availability. Emerging evidence suggests that proteolysis can also be regulated by substrate glycosylation and that glycosylation of individual sites on a substrate can decrease, or in rare cases, increase its sensitivity to proteolysis. In the present study we investigated the relationship between site-specific O-glycosylation and proteolytic cleavage of extracellular proteins. By in silico analysis we found a significant association between O-glycosylation sites and cleavage sites. We then used a positional proteomic strategy, Terminal Amine Isotopic Labelling of Substrates (TAILS) to map in vivo cleavage sites in HepG2 cells with and without one of the key initiating GalNAc-transferases, GalNAc-T2. To reduce complexity, the comparison were performed in so-called SimpleCells. In these cells the Core 1 β3-galactosyltransferase-specific molecular chaperone (COSMC; C1GALT1C1) has been knocked out, resulting in global truncation of O-glycans leaving only the initial GalNAc. Surprisingly, we found that loss of GalNAc-T2 resulted in not only an increase in cleavage, but also a decrease in cleavage across a broad range of other substrates, including key regulators of the protease network. GALNT2 has previously been identified as a candidate gene for dyslipidaemia and has been shown to specifically glycosylate central regulators of lipid homeostasis, here we find altered processing of several of these regulators including Apolipoproteins B (ApoB) and the Phospholipid transfer protein (PLTP), providing new clues to the link between GALNT2 and lipid homeostasis. In contrast to the findings from the majority of previous studies, these results indicate that O-glycosylation can both decrease and increase substrate cleavage. We challenged this system with exogenous matrix metalloproteinase 9 (MMP9) and neutrophil elastase and observed substantial changes to the neutrophil elastase N-terminome, but no evidence for a similar role for MMP9. Overall, we show that loss of O-glycosylation leads to a general decrease in cleavage, that GalNAc-T2 O-glycosylation affects key regulators of the cellular proteolytic network, including multiple members of the Serpin family.

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

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte

SUBMITTER: Sergey Vakhrushev

LAB HEAD: Hans Wandall

PROVIDER: PXD009453 | Pride | 2018-10-08

REPOSITORIES: Pride

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			Action	DRS
	140919_SK_PRETAILS_5_1to1_90min_top10_HCD_2ul-01.msf	Msf
	140919_SK_PRETAILS_5_1to1_90min_top10_HCD_2ul.msf	Msf
	140919_SK_PRETAILS_5_1to1_90min_top10_HCD_2ul.raw	Raw
	140919_SK_PRETAILS_8_1to1_90min_top10_HCD_2ul-01.msf	Msf
	140919_SK_PRETAILS_8_1to1_90min_top10_HCD_2ul.msf	Msf

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TAILS N-terminomics and proteomics reveal complex regulation of proteolytic cleavage by <i>O</i>-glycosylation.

King Sarah L SL Goth Christoffer K CK Eckhard Ulrich U Joshi Hiren J HJ Haue Amalie D AD Vakhrushev Sergey Y SY Schjoldager Katrine T KT Overall Christopher M CM Wandall Hans H HH

The Journal of biological chemistry 20180328 20

Proteolytic processing is an irreversible post-translational modification functioning as a ubiquitous regulator of cellular activity. Protease activity is tightly regulated via control of gene expression, enzyme and substrate compartmentalization, zymogen activation, enzyme inactivation, and substrate availability. Emerging evidence suggests that proteolysis can also be regulated by substrate glycosylation and that glycosylation of individual sites on a substrate can decrease or, in rare cases, ...[more]

PMID: 29593093

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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.