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.

Fig. 1.

Probing isoform-specific functions of GalNAc-Ts by ZFN gene targeting glycosyltransferases. (A) Depiction of the strategy for identification of differential GalNAc-T isoform-specific O-glycoproteomes. (Section 1) ZFN targeting of COSMC in HepG2 (SC) generates cell lines with homogenous and truncated O-glycosylation (GalNAc), and subsequent targeting of GALNT2 (SC/T2^−/−) eliminates GalNAc-T2 nonredundant O-glycosylation allowing for comparative analysis of O-glycoproteomes of isogenic cell lines with and without GALNT2. (Section 2) Proteins from total cell lysates or cell culture supernatant from HepG2-SC or SC/T2^−/− are digested by trypsin. (Section 3) GalNAc-glycopeptides are isolated and separated by lectin weak affinity chromatography. (Section 4) O-glycosylation sites are identified by nLC/MS/MS and comparison between the two cell lines produce candidates for GalNAc-T2–specific contribution. (B) Depiction of Cosmc and GalNAc-T2 protein domains and targeted DNA sequences where introduced mutations are shown. (C) Immunofluorescence staining of isogenic HepG2 cell lines as indicated.

Fig. 2.

GalNAc-T2 controls O-glycosylation of ApoC-III in HepG2 cells. (A) Venn diagram illustrating distribution of identified O-glycoproteins. ApoC-III was identified in HepG2-SC and SC/T2^−/− secretomes and therefore selected for analysis by GalNAc-T enzyme assays. (B) In vitro glycosylation of an ApoC-III peptide substrate as indicated with GalNAc-T1, T2, T4, and T11 monitored by MALDI-TOF analysis (2-h time point shown). Masses of peptides and glycopeptides are shown with the predicted number of incorporated GalNAc residues indicated Left of the peaks. (C) Schematic drawing of ApoC-III indicating the position of O-glycosylated Thr⁹⁴. (D) Immunoprecipitation of ApoC-III from cell culture media of HepG2 wild-type cells, HepG2-T2^−/−, HepG2-T2^−/−/T2^+/+, HepG2-T1^−/−, and 50 ng ApoC-III standard from human serum. SDS/PAGE Western blot with anti–ApoC-III. (E) SDS/PAGE Western blot of 1 μL human serum from three healthy blood donors with anti–ApoC-III. Migration of glycosylated and nonglycosylated ApoC-III is indicated with arrows.

Fig. 3.

Increased PC processing of ANGPTL3 in HepG2-T2^−/− cells. (A) Immunoprecipitation of ANGPTL3 from HepG2 wild-type and T2^−/− cells induced with 5 μM T0901317 for 48 h. SDS/PAGE Western blot with anti-ANGPTL3. Control lane includes 20 ng recombinant nonglycosylated ANGPTL3. Full-length ANGPTL3 migrates to ∼62 kDa and cleaved N-terminal ANGPTL3 migrates to ∼38 kDa as indicated by arrows.