Abstract
O-Linked GlcNAc transferase (OGT) possesses dual glycosyltransferase–protease activities. OGT thereby stably glycosylates serines and threonines of numerous proteins and, via a transient glutamate glycosylation, cleaves a single known substrate—the so-called HCF-1PRO repeat of the transcriptional co-regulator host-cell factor 1 (HCF-1). Here, we probed the relationship between these distinct glycosylation and proteolytic activities. For proteolysis, the HCF-1PRO repeat possesses an important extended threonine-rich region that is tightly bound by the OGT tetratricopeptide-repeat (TPR) region. We report that linkage of this HCF-1PRO-repeat, threonine-rich region to heterologous substrate sequences also potentiates robust serine glycosylation with the otherwise poor Rp-αS-UDP-GlcNAc diastereomer phosphorothioate and UDP-5S-GlcNAc OGT co-substrates. Furthermore, it potentiated proteolysis of a non-HCF-1PRO-repeat cleavage sequence, provided it contained an appropriately positioned glutamate residue. Using serine- or glutamate-containing HCF-1PRO-repeat sequences, we show that proposed OGT-based or UDP-GlcNAc–based serine-acceptor residue activation mechanisms can be circumvented independently, but not when disrupted together. In contrast, disruption of both proposed activation mechanisms even in combination did not inhibit OGT-mediated proteolysis. These results reveal a multiplicity of OGT glycosylation strategies, some leading to proteolysis, which could be targets of alternative molecular regulatory strategies.
Highlights
O-Linked GlcNAc transferase (OGT) possesses dual glycosyltransferase–protease activities
Consistent with previous results using other substrates (5, 9), casein kinase 2 (CK2) substrate O-GlcNAcylation by OGT was maintained, albeit with reduced efficiency, when Sp-␣S-UDPGlcNAc was utilized, whereas no O-GlcNAcylated CK2 protein was observed in the presence of Rp-␣S-UDP-GlcNAc (Fig. 2A, compare lanes 3 and 4)
Similar to these E10S glycosylation results, we have previously shown that the glutamate residue at position 10 (Glu-10) HCF-1PRO-repeat proteolysis substrate is active with either the Rp-␣S-UDP-GlcNAc diastereomer co-substrate or the OGT Swap mutant, but we had not tested them together for proteolysis (9)
Summary
To study the canonical serine and threonine glycosylation and noncanonical glycosyl glutamate–induced cleavage activities of OGT, we used human casein kinase 2 (CK2) and HCF1PRO-repeat substrates, respectively, using both native and recombinant proteins as well as synthetic peptides. For glycosylation, the E10S HCF-1PRO-repeat substrate independently bypasses a requirement for the ␣-phosphate pro-RP oxygen or for the Asp-554 residue implicated in the proposed Grotthuss mechanism but not both in combination Similar to these E10S glycosylation results, we have previously shown that the Glu-10 HCF-1PRO-repeat proteolysis substrate is active with either the Rp-␣S-UDP-GlcNAc diastereomer co-substrate or the OGT Swap mutant, but we had not tested them together for proteolysis (9). In contrast to the E10S serine glycosylation substrate, the Glu-10 substrate is active for proteolysis in the presence of both the Rp-␣S-UDP-GlcNAc diastereomer co-substrate and the OGT Swap mutant (compare lanes [2,3,4,5,6] and 7) Such a result could be explained if proteolysis does not require assisted deprotonation of the target glutamate for its glycosylation and subsequent spontaneous backbone cleavage
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
