Functional expression of O-linked GlcNAc transferase - Domain structure and substrate specificity

O-GlcNAc transferase (OGT) modifies nuclear pore proteins and transcription factors. In Arabidopsis, the OGT homolog participates in the gibberellin s ignaling pathway. We and others have proposed that mammalian OGT is the ter minal step in a glucose-sensitive signal transduction pathway that becomes disregulated in insulin resistance. To facilitate mutational analysis of OG T in the absence of competing endogenous activity, we expressed the 103-kDa human. OGT in Escherichia coli. Kinetic parameters for the purified recomb inant enzyme (K-m = 1.2 mu M for Nup 62; K-m = 0.5 mu M for UDP-GlcNAc) are nearly identical to purified mammalian OGT. Deletions in the highly conser ved C terminus result in a complete loss of activity. The N-terminal tetrat ricopeptide repeat domain is required for optimal recognition of substrates , Removal of the first three tetratricopeptide repeats greatly reduces the O-GlcNAc addition to macromolecular substrates. However, this altered enzym e retains full activity against appropriate synthetic peptides. Autoglycosy lation of OGT is augmented when the first six tetratricopeptide repeats are removed showing that these repeats are not required for catalysis. Given i ts proposed role in modulating insulin action, OGT may modify kinases invol ved in this signaling cascade. Among the many kinases tested, OGT glycosyla tes glycogen synthase kinase-3 and casein kinase II, two enzymes critical i n the regulation of glycogen synthesis.