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.
Highlights
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
Whereas it seems logical that an increase in the amount of a glycosyltransferase enzyme in a cell should lead to an increase in glycosylation efficiency, this may not be the case given the complex and intertwined pathways of glycan assembly, where multiple enzymes utilize the same substrates, and isoenzymes have overlapping functions but potentially different subcellular localizations
We chose to study the effects of enzyme dose in the context of the large family of GalNAc-T isoenzymes, especially because several of these enzymes appear to be involved in human disorders through dysregulation of
Summary
HEK cells express a subset of the 20 GALNT genes with the major isoenzymes expressed being GALNT1 and -T2; with lower levels of GALNT3, -T7, -T11, and -T18; and with barely detectable levels of GALNT6 and -T16 (Fig. S1). Glycopeptides from nine glycoproteins were induced at 4 ng/ml (green in Fig. 6 and Data S1C), and among these was a glycopeptide from GalNAc-T2 itself 55DLHHSNGEEK64 (unambiguous glycosites in boldface type) This glycosite in GalNAc-T2 is not a specific substrate for the enzyme, and we interpret that this finding reflects induction of the enzyme protein and subsequent glycosylation by the other endogenous GalNAc-Ts expressed in HEK cells (Fig. S1). We interpret the data to demonstrate that induction of GalNAc-T2 and -T11 in HEK cells produces discrete glycosylation of distinct O-glycosites in a tight and dosedependent manner without affecting the major part of the O-glycoproteome, which is covered by functional redundancy among GalNAc-T isoforms, even though many of the unaffected sites serve as substrates for the induced enzymes in in vitro assays [21]
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