Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines

J Biol Chem. 2018 Dec 7;293(49):19064-19077. doi: 10.1074/jbc.RA118.004516. Epub 2018 Oct 16.

Abstract

The GalNAc-type O-glycoproteome is orchestrated by a large family of polypeptide GalNAc-transferase isoenzymes (GalNAc-Ts) with partially overlapping contributions to the O-glycoproteome besides distinct nonredundant functions. Increasing evidence indicates that individual GalNAc-Ts co-regulate and fine-tune specific protein functions in health and disease, and deficiencies in individual GALNT genes underlie congenital diseases with distinct phenotypes. Studies of GalNAc-T specificities have mainly been performed with in vitro enzyme assays using short peptide substrates, but recently quantitative differential O-glycoproteomics of isogenic cells with and without GALNT genes has enabled a more unbiased exploration of the nonredundant contributions of individual GalNAc-Ts. Both approaches suggest that fairly small subsets of O-glycosites are nonredundantly regulated by specific GalNAc-Ts, but how these isoenzymes orchestrate regulation among competing redundant substrates is unclear. To explore this, here we developed isogenic cell model systems with Tet-On inducible expression of two GalNAc-T genes, GALNT2 and GALNT11, in a knockout background in HEK293 cells. Using quantitative O-glycoproteomics with tandem-mass-tag (TMT) labeling, we found that isoform-specific glycosites are glycosylated in a dose-dependent manner and that induction of GalNAc-T2 or -T11 produces discrete glycosylation effects without affecting the major part of the O-glycoproteome. These results support previous findings indicating that individual GalNAc-T isoenzymes can serve in fine-tuned regulation of distinct protein functions.

Keywords: GalNAc-transferase; O-GalNAc; O-glycosylation; cellular regulation; glycan; glycobiology; glycomics; glycoproteomics; glycosylation; glycosyltransferase; inducible expression; mass spectrometry (MS); mucin type; protein glycosylation; tandem mass tag.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Glycosylation
  • HEK293 Cells
  • Humans
  • Isoenzymes / metabolism
  • N-Acetylgalactosaminyltransferases / metabolism*
  • Polypeptide N-acetylgalactosaminyltransferase
  • Proteome / metabolism*
  • Proteomics / methods

Substances

  • Isoenzymes
  • Proteome
  • GALNT11 protein, human
  • N-Acetylgalactosaminyltransferases