Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology

Catharina Steentoft, Sergey Vakhrushev, Hiren Jitendra Joshi, Yun Kong, Malene B Vester-Christensen, Katrine T-BG Schjoldager, Kirstine Lavrsen, Sally Anne Malene Dabelsteen, Nis Borbye Pedersen, Lara Marcos-Silva, Ramneek Gupta, Eric Paul Bennett, Ulla Mandel, Søren Brunak, Hans H. Wandall, Steven Bruce Levery, Henrik Clausen

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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.
Original languageEnglish
JournalEMBO Journal
Volume32
Issue number10
Pages (from-to)1478-1488
ISSN0261-4189
DOIs
Publication statusPublished - 2013

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