title: A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation.
abstract: Regulated shedding of the ectodomain of cell membrane proteins by proteases is a common process that releases the extracellular domain from the cell and activates cell signaling. Ectodomain shedding occurs in the immediate extracellular juxtamembrane region, which is also where O-glycosylation is often found and examples of crosstalk between shedding and O-glycosylation have been reported. Here, we systematically investigated the potential of site-specific O-glycosylation mediated by distinct polypeptide GalNAc-transferase (GalNAc-T) isoforms to coregulate ectodomain shedding mediated by the A Disintegrin And Metalloproteinase (ADAM) subfamily of proteases and in particular ADAM17. We analyzed 25 membrane proteins that are known to undergo ADAM17 shedding and where the processing sites included Ser/Thr residues within ± 4 residues that could represent O-glycosites. We used in vitro GalNAc-T enzyme and ADAM cleavage assays to demonstrate that shedding of at least 12 of these proteins are potentially coregulated by O-glycosylation. Using TNF-α as an example, we confirmed that shedding mediated by ADAM17 is coregulated by O-glycosylation controlled by the GalNAc-T2 isoform both ex vivo in isogenic cell models and in vivo in mouse Galnt2 knockouts. The study provides compelling evidence for a wider role of site-specific O-glycosylation in ectodomain shedding.
authors.affiliation: Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark;
authors.forename: Christoffer K
authors.initials: CK
authors.lastname: Goth
authors.affiliation: Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark;
authors.forename: Adnan
authors.initials: A
authors.lastname: Halim
authors.affiliation: Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
authors.forename: Sumeet A
authors.initials: SA
authors.lastname: Khetarpal
authors.affiliation: Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Rader
authors.affiliation: Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark;
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
authors.affiliation: Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; Schjoldager@sund.ku.dk.
authors.forename: Katrine T-B G
authors.initials: KT
authors.lastname: Schjoldager
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D017930:Genes, Reporter; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000097:blood / Q000378:metabolism; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D051722:ADAM Proteins; D006020:Glycopeptides; D008070:Lipopolysaccharides; D017350:N-Acetylgalactosaminyltransferases; D014409:Tumor Necrosis Factor-alpha; D000097763:Polypeptide N-acetylgalactosaminyltransferase
keywords: ACE; ADAM10; ErbB4; IL-6RA; TACE