The conserved threonine-rich region of the HCF-1PRO repeat activates promiscuous OGT: UDP-GlcNAc glycosylation and proteolysis activities

Vaibhav Kapuria, Ute F. Röhrig, Patrice Waridel, Fabienne Lammers, Vladimir S. Borodkin, Daan M. F. van Aalten, Vincent Zoete, Winship Herr (Lead / Corresponding author)

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    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-1 PRO 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-1 PRO 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-1 PRO-repeat, threonine-rich region to heterologous substrate sequences also potentiates robust serine glycosylation with the otherwise poor R p-S-UDP-GlcNAc diastereomer phosphorothioate and UDP-5S-GlcNAc OGT co-substrates. Furthermore, it potentiated proteolysis of a non-HCF-1 PRO-repeat cleavage sequence, provided it contained an appropriately positioned glutamate residue. Using serine- or glutamate-containing HCF-1 PRO-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.

    Original languageEnglish
    Pages (from-to)17754-17768
    Number of pages15
    JournalJournal of Biological Chemistry
    Volume293
    Issue number46
    Early online date17 Sept 2018
    DOIs
    Publication statusPublished - 16 Nov 2018

    Keywords

    • Host-cell factor-1
    • enzyme mechanism
    • glycobiology
    • post-translational modification (PTM)
    • O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT)
    • O-GlcNAcylation

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