A metabolic labeling approach for glycoproteomic analysis reveals altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells.
Epithelial ovarian cancer (EOC) is a disease responsible for more deaths among women in the Western world than all other gynecologic malignancies. There is urgent need for new therapeutic targets and a better understanding of EOC initiation and progression. We have previously identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene, a member of the GalNAc-transferases (GalNAc-Ts) gene family, as hypomethylated and overexpressed in high-grade serous EOC tumors, compared to low malignant potential EOC tumors and normal ovarian tissues. This data also suggested for a role of GALNT3 in aberrant EOC glycosylation, possibly implicated in disease progression. To evaluate differential glycosylation in EOC caused by modulations in GALNT3 expression, we used a metabolic labeling strategy for enrichment and mass spectrometry-based characterization of glycoproteins following GALNT3 gene knockdown (KD) in A2780s EOC cells. A total of 589 differentially expressed glycoproteins were identified upon GALNT3 KD. Most identified proteins were involved in mechanisms of cellular metabolic functions, post-translational modifications, and some have been reported to be implicated in EOC etiology. The GALNT3-dependent glycoproteins identified by this metabolic labeling approach support the oncogenic role of GALNT3 in EOC dissemination and may be pursued as novel EOC biomarkers and/or therapeutic targets.



Knowledge of the O-glycoproteome has been relatively elusive, and the functions of the individual polypeptide GalNAc-Ts have been poorly characterized. Alterations in GalNAc-Ts expression were shown to provide huge variability in the O-glycoproteome in various pathologies, including cancer. The application of a chemical biology approach for the metabolic labeling and subsequent characterization of O-glycoproteins in EOC using the Ac4GalNAz metabolite has provided a strategy allowing for proteomic discovery of GalNAc-Ts specific functions. Our study supports an essential role of one of the GalNAc-Ts - GALNT3, in EOC dissemination, including its implication in modulating PTMs and EOC metabolism. Our approach validates the use of the applied metabolic strategy to identify important functions of GalNAc-Ts in normal and pathological conditions.
Department of Molecular Medicine, Laval University, Québec, PQ, Canada; Centre de recherche du CHU de Québec, L'Hôtel-Dieu de Québec, Québec, PQ, Canada.
Razan
R
Sheta
Department of Chemistry, Stanford University, Stanford, CA, USA.
Christina M
CM
Woo
Centre de recherche du CHU de Québec, CHUL, Québec, PQ, Canada.
Florence
F
Roux-Dalvai
Centre de recherche du CHU de Québec, CHUL, Québec, PQ, Canada.
Frédéric
F
Fournier
Centre de recherche du CHU de Québec, CHUL, Québec, PQ, Canada.
Sylvie
S
Bourassa
Department of Molecular Medicine, Laval University, Québec, PQ, Canada; Centre de recherche du CHU de Québec, CHUL, Québec, PQ, Canada.
Arnaud
A
Droit
Department of Chemistry, Stanford University, Stanford, CA, USA; Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA.
Carolyn R
CR
Bertozzi
Department of Molecular Medicine, Laval University, Québec, PQ, Canada; Centre de recherche du CHU de Québec, L'Hôtel-Dieu de Québec, Québec, PQ, Canada.
Dimcho
D
Bachvarov
D005260:Female; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D006023:Glycoproteins / Q000032:analysis* / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics*; D010051:Ovarian Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D006023:Glycoproteins; D017350:N-Acetylgalactosaminyltransferases; D000097763:Polypeptide N-acetylgalactosaminyltransferase
Epithelial ovarian cancer; GALNT3; Glycoproteomics; Glycosylation; Metabolic labeling; Pathway and network analysis