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

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 coregulate and fine-tune specific protein functions in health and disease, and deficiencies in individual GALNT genes underlie congenital diseases with distinct phenotypes. Studies of Gal-NAc-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 isoformspecific 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.

Metabolic precision labeling enables selective probing of O-linkedN-acetylgalactosamine glycosylation

“…Studying O-GalNAc glycoproteins by MS-based glycoproteomics is complicated by glycan heterogeneity, the lack of O-glycosylation consensus sequences, and selective enrichment tools. Further complexity is added by the interplay of 20 GalNAc transferase (GalNAc-T1…T20) isoenzymes that mediate the first O-GalNAc biosynthesis step (22, 23). In a “bump-and-hole” (BH) approach, we have recently engineered GalNAc-Ts to carry a double mutation to preferentially accept UDP-GalNAc analogs with bulky chemical, editable tags (24, 25).…”

Genetic Variation Regulates Opioid-Induced Respiratory Depression in Mice

“…The non-synonymous serine-to-leucine SNP changes a conserved residue within the Ricin B lectin domain of GALNT11 and would be predicted to impede O-linked glycosylation. GALTN11 has been shown to uniquely glycosylate at least 313 glycoproteins in HEK cells 33 . Using the Jaccard similarity tool in the GeneWeaver 34 system for integrative functional genomics, we determined that of the 313 human glycopeptides, 287 mouse orthologs are expressed in the mouse pre-Bötzinger complex 32 (Table S2).…”

“…In order to assess the functional sufficiency of Galnt11 as a candidate gene the literature was searched to identify genome-wide studies characterizing glycosylation targets of GALTN11, one study was identified 33 and these genes were added to the GeneWeaver Database (GS356053). Using the Jaccard similarity tool, we overlapped the glycosylation targets with the genes expressed in the mouse pre-Bötzinger complex.…”

Chemical precision glyco-mutagenesis by glycosyltransferase engineering in living cells

“…Knock-outs of single GalNAcTs in the SimpleCell background have been profiled by glycosproteomics (18,21). Similarly, glycopeptides from non-SimpleCells with titratable GalNAcT knock-in have been enzymatically simplified to allow for enrichment and MS (22).…”

“…These studies have revealed comprehensive glycoproteomics datasets, but suffer from loss of 20 glycan elaboration and laborious genome engineering required for targeted knock-in. Further, the activity of GalNAcT isoenzymes is both redundant and competitive, such that compensation and/or 4 shift of glycosylation sites occurs upon knock-out (21,22). A gain-of-function strategy to visualize the products of a particular GT on the living cell is currently elusive.…”