Ser and Thr acceptor preferences of the GalNAc-Ts vary among isoenzymes to modulate mucin-type O-glycosylation

Abstract

A family of polypeptide GalNAc-transferases (GalNAc-Ts) initiates mucin-type O-glycosylation, transferring GalNAc onto hydroxyl groups of Ser and Thr residues of target substrates. The 20 GalNAc-T isoenzymes in humans are classified into nine subfamilies according to sequence similarity. GalNAc-Ts select their sites of glycosylation based on weak and overlapping peptide sequence motifs, as well prior substrate O-GalNAc glycosylation at sites both remote (long-range) and neighboring (short-range) the acceptor. Together, these preferences vary among GalNAc-Ts imparting each isoenzyme with its own unique specificity. Studies on the first identified GalNAc-Ts showed Thr acceptors were preferred over Ser acceptors; however studies comparing Thr vs. Ser glycosylation across the GalNAc-T family are lacking. Using a series of identical random peptide substrates, with single Thr or Ser acceptor sites, we determined the rate differences (Thr/Ser rate ratio) between Thr and Ser substrate glycosylation for 12 isoenzymes (representing 7 GalNAc-T subfamilies). These Thr/Ser rate ratios varied across subfamilies, ranging from ~2 to ~18 (for GalNAc-T4/GalNAc-T12 and GalNAc-T3/GalNAc-T6, respectively), while nearly identical Thr/Ser rate ratios were observed for isoenzymes within subfamilies. Furthermore, the Thr/Ser rate ratios did not appreciably vary over a series of fixed sequence substrates of different relative activities, suggesting the ratio is a constant for each isoenzyme against single acceptor substrates. Finally, based on GalNAc-T structures, the different Thr/Ser rate ratios likely reflect differences in the strengths of the Thr acceptor methyl group binding to the active site pocket. With this work, another activity that further differentiates substrate specificity among the GalNAc-Ts has been identified.

Exploring the glycosylation of mucins by use of O-glycodomain reporters recombinantly expressed in glycoengineered HEK293 cells

“…More likely, the lower occupancy for the MUC20 and MUC21 TR domains is related to a greater diversity in amino acid usage and a higher use of Ser than Thr residues in these TRs compared with what is found in secreted mucin TRs. The sequence context of O-glycosites clearly affects O-glycosylation, and Ser O-glycosites are considered poorer acceptor substrates than Thr sites for GALNTs ( 49 ), and GalNAc residues attached to Ser and Thr residues attain distinct conformations ( 50 ). We are currently not able to analyze TR reporters expressed as membrane-bound proteins.…”

Putative linear motifs mediate the trafficking to apical and basolateral membranes

“…Accumulation of S/T rich motifs is likely connected with mucin-type O-glycosylation phenomena intimately connected to a preferentially apical sorting [21,[23][24]. These sequences (especially due to their prominent Thr and Pro content) closely resemble the target sites of O-GalNac transferases [25][26]. However, due to the complex, processive nature of GalNac-T enzymes, the exact sequence of O-glycosylation sites is impossible to predict [27][28].…”

The Role of APP O-Glycosylation in Alzheimer’s Disease

“…Substrate preference varies among isozymes [ 30 , 31 , 32 ]. Both Ser and Thr residues can be an acceptor, and a number of ppGalNAc-Ts exhibit a preference for Thr over Ser [ 33 ]. Using randomized peptide substrates, differences in Thr and Ser substrate glycosylation rates were determined.…”