Ser and Thr Acceptor Preferences of the GalNAc-Ts Vary Among Isoenzymes to Modulate Mucin Type O-Glycosylation

Ser and Thr Acceptor Preferences of the GalNAc-Ts Vary Among Isoenzymes to Modulate Mucin Type O-Glycosylation. Daniel EJP, Las Rivas M, Lira-Navarrete E, García-García A, Hurtado-Guerrero R, Clausen H, Gerken TA. Glycobiology. 2020 Apr 18:cwaa036. doi: 10.1093/glycob/cwaa036. Online ahead of print. PMID: 32304323

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 9 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 versus 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/T12 and GalNAc-T3/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.

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