title: Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology.
abstract: Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc-type O-glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc-transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O-glycosylation (SimpleCells) that enables proteome-wide discovery of O-glycan sites using 'bottom-up' ETD-based mass spectrometric analysis. We implemented this on 12 human cell lines from different organs, and present a first map of the human O-glycoproteome with almost 3000 glycosites in over 600 O-glycoproteins as well as an improved NetOGlyc4.0 model for prediction of O-glycosylation. The finding of unique subsets of O-glycoproteins in each cell line provides evidence that the O-glycoproteome is differentially regulated and dynamic. The greatly expanded view of the O-glycoproteome should facilitate the exploration of how site-specific O-glycosylation regulates protein function.
authors.affiliation: Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine and School of Dentistry, University of Copenhagen, Copenhagen N, Denmark.
authors.forename: Catharina
authors.initials: C
authors.lastname: Steentoft
authors.forename: Sergey Y
authors.initials: SY
authors.lastname: Vakhrushev
authors.forename: Hiren J
authors.initials: HJ
authors.lastname: Joshi
authors.forename: Yun
authors.initials: Y
authors.lastname: Kong
authors.forename: Malene B
authors.initials: MB
authors.lastname: Vester-Christensen
authors.forename: Katrine T-B G
authors.initials: KT
authors.lastname: Schjoldager
authors.forename: Kirstine
authors.initials: K
authors.lastname: Lavrsen
authors.forename: Sally
authors.initials: S
authors.lastname: Dabelsteen
authors.forename: Nis B
authors.initials: NB
authors.lastname: Pedersen
authors.forename: Lara
authors.initials: L
authors.lastname: Marcos-Silva
authors.forename: Ramneek
authors.initials: R
authors.lastname: Gupta
authors.forename: Eric Paul
authors.initials: EP
authors.lastname: Bennett
authors.forename: Ulla
authors.initials: U
authors.lastname: Mandel
authors.forename: Søren
authors.initials: S
authors.lastname: Brunak
authors.forename: Hans H
authors.initials: HH
authors.lastname: Wandall
authors.forename: Steven B
authors.initials: SB
authors.lastname: Levery
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
mesh_terms: D000465:Algorithms; D020816:Amino Acid Motifs; D045744:Cell Line, Tumor; D005818:Genetic Engineering / Q000379:methods; D006023:Glycoproteins / Q000032:analysis* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D006023:Glycoproteins; D017350:N-Acetylgalactosaminyltransferases; D000097763:Polypeptide N-acetylgalactosaminyltransferase