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TMT10-TAILS investigation of the proteolytic landscape in B cell activation

ABSTRACT: The N-terminome in EBV-B cells from a human patient homozygous for an W580S mutation in MALT1 were analyzed by TMT10-plex TAILS and compared to EBV-B cells from heterozygous subjects under resting and PMA/ionomycin stimulation for 2 and 4 h.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Christopher M. Overall

PROVIDER: MSV000083027 | MassIVE | Thu Oct 11 16:31:00 BST 2018

SECONDARY ACCESSION(S): PXD006723

REPOSITORIES: MassIVE

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Project description: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.

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TMT10-TAILS investigation of the proteolytic landscape in B cell activation

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