Abstract
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.
Highlights
PpGalNAc transferases, which initiate O-glycosylation, possess a poorly understood lectin domain
Numerous studies on the ppGalNAc Ts impart substrate specificity to nominally nonspecific catalytic have demonstrated that their lectin domains serve similar functions to modulate glycopeptide substrate recognition and specificity (48 –53), but to date there has been no systematic study of the family against a common set ofpeptide substrates
Our findings have unambiguously revealed that prior GalNAc-OThr(Ser) substrate glycosylation can be recognized by these transferases in a specific N- or C-terminal direction that varies with ppGalNAc T isoform
Summary
Cated in the above studies, little is known about the actual site(s) of glycosylation and their specificity, and even less is known of the actual molecular mechanism(s) leading to their biological function. Our recent studies utilizing short random (glyco)peptide substrates have shown that the ppGalNAc Ts possess specific binding preferences that vary among isoforms for peptide and even GalNAc-O-Ser/Thrcontaining glycopeptide substrates [43,44,45]. We and others have shown that the placement of a neighboring GalNAc-O-Ser/Thr residue near a glycosylation site produces a range of transferase-specific effects (i.e. a relative inhibition of glycosylation (i.e. ppGalNAc T1 and T2 [46, 47]), an alteration or shift in glycosylation site (ppGalNAc T2 and T4 (48 –51)), or even a large apparent rate enhancement (i.e. requirement for glycosylated substrate) (ppGalNAc T7 and T10 [45, 51,52,53]). PpGalNAc T glycopeptide substrate utilization, we have extended our studies to the function of the lectin domain, utilizing a series of random glycopeptides containing N- or C- terminally placed GalNAc-O-Thr residues. Because of the large differences in glycosylation rates observed for some isoforms between an N- or C-terminally placed GalNAc-O-Thr, we believe we have uncovered another level of control of mucin type O-glycosylation that will further advance our understanding of the regulation of this important modification
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