TY - JOUR

T1 - TAILS N-Terminomics and proteomics reveal complex regulation of proteolytic cleavage by O-glycosylation

AU - King, Sarah L

AU - Goth, Christoffer K

AU - Eckhard, Ulrich

AU - Joshi, Hiren Jitendra

AU - Haue, Amalie D

AU - Vakhrushev, Sergey

AU - Schjoldager, Katrine

AU - Overall, Christopher M

AU - Wandall, Hans H

N1 - Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

PY - 2018

Y1 - 2018

N2 - 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, increase its sensitivity to proteolysis. Here, we investigated the relationship between site-specific, mucin-type (or GalNAc-type) O-glycosylation and proteolytic cleavage of extracellular proteins. Using in silico analysis, we found that O-glycosylation and cleavage sites are significantly associated with each other. We then used a positional proteomic strategy, Terminal Amine Isotopic Labeling of Substrates (TAILS), to map the in vivo cleavage sites in HepG2 SimpleCells with and without one of the key initiating GalNAc transferases, GalNAc-T2, and after treatment with exogenous matrix metalloproteinase 9 (MMP9) or neutrophil elastase. Surprisingly, we found that loss of GalNAc-T2 not only increased cleavage, but also decreased cleavage across a broad range of other substrates, including key regulators of the protease network. We also found altered processing of several central regulators of lipid homeostasis, including apolipoprotein B (ApoB) and the phospholipid transfer protein (PLTP), providing new clues to the previously reported link between GALNT2 and lipid homeostasis. In summary, we show that loss of GalNAc-T2 O-glycosylation leads to a general decrease in cleavage, and that GalNAc-T2 O-glycosylation affects key regulators of the cellular proteolytic network, including multiple members of the Serpin family.

AB - 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, increase its sensitivity to proteolysis. Here, we investigated the relationship between site-specific, mucin-type (or GalNAc-type) O-glycosylation and proteolytic cleavage of extracellular proteins. Using in silico analysis, we found that O-glycosylation and cleavage sites are significantly associated with each other. We then used a positional proteomic strategy, Terminal Amine Isotopic Labeling of Substrates (TAILS), to map the in vivo cleavage sites in HepG2 SimpleCells with and without one of the key initiating GalNAc transferases, GalNAc-T2, and after treatment with exogenous matrix metalloproteinase 9 (MMP9) or neutrophil elastase. Surprisingly, we found that loss of GalNAc-T2 not only increased cleavage, but also decreased cleavage across a broad range of other substrates, including key regulators of the protease network. We also found altered processing of several central regulators of lipid homeostasis, including apolipoprotein B (ApoB) and the phospholipid transfer protein (PLTP), providing new clues to the previously reported link between GALNT2 and lipid homeostasis. In summary, we show that loss of GalNAc-T2 O-glycosylation leads to a general decrease in cleavage, and that GalNAc-T2 O-glycosylation affects key regulators of the cellular proteolytic network, including multiple members of the Serpin family.

U2 - 10.1074/jbc.RA118.001978

DO - 10.1074/jbc.RA118.001978

M3 - Journal article

C2 - 29593093

VL - 293

SP - 7629

EP - 7644

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 20

ER -