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

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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 Simple-Cells, 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 targetingwith Simple- Cells 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.

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Schjoldager, K. T. B. G., Vakhrushev, S. Y., Kong, Y., Steentoft, C., Nudelman, A. S., Pedersen, N. B., … Clausen, H. (2012). Probing isoform-specific functions of polypeptide GalNAc-transferases using zinc finger nuclease glycoengineered SimpleCells. Proceedings of the National Academy of Sciences of the United States of America, 109(25), 9893–9898. https://doi.org/10.1073/pnas.1203563109

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