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 Oglycan 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 Oglycosylation 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.apolipoproteins | angiopoietin-like proteins | genetic engineering | glycoproteins T he GALNT2 gene involved in O-glycosylation has been associated with aberrant serum levels of triglyceride (TG) and highdensity lipoprotein cholesterol (HDL-C) by several genome-wide association studies (GWAS) (1, 2). A direct functional role of this gene was obtained by transient knockdown and overexpression of galnt2 in mouse liver, which resulted in increased and lowered plasma HDL-C, respectively (3). GALNT2 is a member of the largest family of homologous glycosyltransferase isoforms (up to 20) catalyzing the same glycosidic linkage (GalNAcα1-O-Ser/Thr) and initiating protein O-glycosylation (4, 5). These isoforms have overlapping but distinct peptide substrate specificities and identifying essential unique functions for individual GalNAc-T isoforms has been notoriously difficult. In search of putative functions of GALNT2 in lipid metabolism, we previously screened a number of known and predicted O-glycoproteins with roles in lipid metabolism for O-glycosylation by the encoded GalNAc-T2 enzyme and identified ANGPTL3 (6). We found that site-specific O-glycosylation of a Thr residue adjacent to the proprotein convertase (PC) processing site in ANGPTL3 that activates this lipase inhibitor was performed only by the GalNAc-T2 isoform. More recently, a heterozygous mutation in GALNT2 resulting in slightly reduced kinetic properties of the encoded GalNAc-T2 enzyme was found in two probands with elevated HDL and reduced TG (7). ...

Development of Isoform-specific Sensors of Polypeptide GalNAc-transferase Activity

“…Cell Culture-HEK cells, including HEK293 controls and HEK cells deleted for T2 or T3 3 produced by zinc finger nuclease-mediated knock-out similarly to previously described (24,25), were cultured in minimal essential medium (Thermo Scientific, Waltham, MA) with 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA) and 100 IU/ml of penicillin/ streptomycin (Sigma). The cells were transfected with the constructs using the JetPEI transfection reagent (VWR International, Radnor, PA) exactly as described by the manufacture's instructions.…”

“…The chosen 219 KPRAPRTTPF 228 sequence of ANGPTL3 is an in vitro target of T2 and to a lesser extent T3 (26). In liver cells, which naturally express ANGPTL3 and lack T3, the sequence is a specific target of T2 (25) indicating that other family members expressed in these cells do not recognize it. Glycosylation of the sequence on Thr 226 blocks furin-like proprotein processing cleavage at Arg 224 (1,26).…”

Moving the O-glycoproteome from form to function

“…Genome-wide association studies are beginning to reveal unexpected relationships between glycosyltransferases and diseases. In the case of ppGalNAcTs, it will now be possible to identify candidate glycoproteins for further analysis as has been done in this study (1). Strategies such as this are moving the field of glycobiology beyond structural studies and mapping to enhancing our understanding of the functional significance of these complex structures and their relevance to human health.…”

“…In PNAS, Schjoldager et al (1) provide an elegant comeback by using a unique strategy for mapping the location of OGalNAc-linked carbohydrate structures to show how the addition of GalNAc to specific Ser and Thr residues by one of 20 polypeptide GalNAc-transferases (ppGalNAc-Ts) can modulate the biologic activity of the modified glycoprotein. The consequences can be dramatic, for example, altering cholesterol or triglyceride metabolism and thus exerting a significant effect on human health parameters.…”

Not so sweet malignant transformation

“…The ground-breaking work by Radhakrishnan et al (4) in PNAS has taken advantage of a novel technique they developed to identify sites of O-glycan addition to proteins (5,6), to provide evidence that truncation of O-glycans induces oncogenic features, such as enhanced growth and invasion. The results are remarkable not only because they indicate that O-glycans play a critical role in regulating growth and adhesive properties of cells, but because they also demonstrate that multiple different properties associated with an enhanced malignant phenotype are affected when O-glycans are truncated.…”