title: Probing isoform-specific functions of polypeptide GalNAc-transferases using zinc finger nuclease glycoengineered SimpleCells.
abstract: Our knowledge of the O-glycoproteome [N-acetylgalactosamine (GalNAc) type] is highly limited. The O-glycoproteome is differentially regulated in cells by dynamic expression of a subset of 20 polypeptide GalNAc-transferases (GalNAc-Ts), and methods to identify important functions of individual GalNAc-Ts are largely unavailable. We recently introduced SimpleCells, i.e., human cell lines made deficient in O-glycan extension by zinc finger nuclease targeting of a key gene in O-glycan elongation (Cosmc), which allows for proteome-wide discovery of O-glycoproteins. Here we have extended the SimpleCell concept to include proteome-wide discovery of unique functions of individual GalNAc-Ts. We used the GalNAc-T2 isoform implicated in dyslipidemia and the human HepG2 liver cell line to demonstrate unique functions of this isoform. We confirm that GalNAc-T2-directed site-specific O-glycosylation inhibits proprotein activation of the lipase inhibitor ANGPTL3 in HepG2 cells and further identify eight O-glycoproteins exclusively glycosylated by T2 of which one, ApoC-III, is implicated in dyslipidemia. Our study supports an essential role for GalNAc-T2 in lipid metabolism, provides serum biomarkers for GalNAc-T2 enzyme function, and validates the use of GALNT gene targeting with SimpleCells for broad discovery of disease-causing deficiencies in O-glycosylation. The presented glycoengineering strategy opens the way for proteome-wide discovery of functions of GalNAc-T isoforms and their role in congenital diseases and disorders.
authors.affiliation: Copenhagen Center for Glycomics, Department of Cellular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
authors.forename: Katrine T-B G
authors.initials: KT
authors.lastname: Schjoldager
authors.forename: Sergey Y
authors.initials: SY
authors.lastname: Vakhrushev
authors.forename: Yun
authors.initials: Y
authors.lastname: Kong
authors.forename: Catharina
authors.initials: C
authors.lastname: Steentoft
authors.forename: Aaron S
authors.initials: AS
authors.lastname: Nudelman
authors.forename: Nis B
authors.initials: NB
authors.lastname: Pedersen
authors.forename: Hans H
authors.initials: HH
authors.lastname: Wandall
authors.forename: Ulla
authors.initials: U
authors.lastname: Mandel
authors.forename: Eric P
authors.initials: EP
authors.lastname: Bennett
authors.forename: Steven B
authors.initials: SB
authors.lastname: Levery
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
mesh_terms: D000116:Acetylgalactosamine / Q000737:chemistry*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry*; D015335:Molecular Probes; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry / Q000378:metabolism; D020543:Proteome; D053719:Tandem Mass Spectrometry; D016335:Zinc Fingers
chemical_list: D007527:Isoenzymes; D015335:Molecular Probes; D020543:Proteome; D017350:N-Acetylgalactosaminyltransferases; D000116:Acetylgalactosamine