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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.

SUBMITTER: Schjoldager KT 

PROVIDER: S-EPMC3382506 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Probing isoform-specific functions of polypeptide GalNAc-transferases using zinc finger nuclease glycoengineered SimpleCells.

Schjoldager Katrine T-B G KT   Vakhrushev Sergey Y SY   Kong Yun Y   Steentoft Catharina C   Nudelman Aaron S AS   Pedersen Nis B NB   Wandall Hans H HH   Mandel Ulla U   Bennett Eric P EP   Levery Steven B SB   Clausen Henrik H  

Proceedings of the National Academy of Sciences of the United States of America 20120507 25


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 (Cos  ...[more]

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