The Lectin Domain of the Polypeptide GalNAc Transferase Family of Glycosyltransferases (ppGalNAc Ts) Acts as a Switch Directing Glycopeptide Substrate Glycosylation in an N- or C-terminal Direction, Further Controlling Mucin Type O-Glycosylation

Mucin type O-glycosylation is initiated by a large family of polypeptide GalNAc transferases (ppGalNAc Ts) that add α-GalNAc to the Ser and Thr residues of peptides. Of the 20 human isoforms, all but one are composed of two globular domains linked by a short flexible linker: a catalytic domain and a ricin-like lectin carbohydrate binding domain. Presently, the roles of the catalytic and lectin domains in peptide and glycopeptide recognition and specificity remain unclear. To systematically study the role of the lectin domain in ppGalNAc T glycopeptide substrate utilization, we have developed a series of novel random glycopeptide substrates containing a single GalNAc-O-Thr residue placed near either the N or C terminus of the glycopeptide substrate. Our results reveal that the presence and N- or C-terminal placement of the GalNAc-O-Thr can be important determinants of overall catalytic activity and specificity that differ between transferase isoforms. For example, ppGalNAc T1, T2, and T14 prefer C-terminally placed GalNAc-O-Thr, whereas ppGalNAc T3 and T6 prefer N-terminally placed GalNAc-O-Thr. Several transferase isoforms, ppGalNAc T5, T13, and T16, display equally enhanced N- or C-terminal activities relative to the nonglycosylated control peptides. This N- and/or C-terminal selectivity is presumably due to weak glycopeptide binding to the lectin domain, whose orientation relative to the catalytic domain is dynamic and isoform-dependent. Such N- or C-terminal glycopeptide selectivity provides an additional level of control or fidelity for the O-glycosylation of biologically significant sites and suggests that O-glycosylation may in some instances be exquisitely controlled. Background: ppGalNAc transferases, which initiate O-glycosylation, possess a poorly understood lectin domain. Results: The lectin domain directs glycosylation in an N- or C- terminal direction in an isoform-specific manner. Conclusion: Unanticipated isoform-specific directionality was revealed for modification of glycopeptide substrates. Significance: A novel mechanism of controlling of mucin type O-glycosylation has been discovered based on tethered lectin domains specifying N- or C-terminal modification of glycopeptide substrates.