Generation of Properly Folded Epidermal Growth Factor-Like (EGF) Repeats and Glycosyltransferases Enables In Vitro O-Glycosylation.

The epidermal growth factor-like (EGF) domain is an evolutionarily conserved motif found widely distributed among numerous secreted and membrane-anchored proteins, including the Notch receptors. Notch receptors include numerous EGF repeats tandemly connected in the extracellular domain. These EGF repeats must be properly folded in order for them to undergo the three different types of O-glycosylation associated with these extracellular proteins: O-fucose, O-glucose, and O-N-acetylglucosamine via glycosyltransferases POFUT1, POGLUT1, and EOGT. The O-glycosylation of the EGF repeats in the Notch receptors regulates the activation of Notch signaling and mutations in POFUT1, POGLUT1, and EOGT have been linked to specific human diseases. A large amount of EGF repeat and glycosyltransferase protein is required to construct an in vitro O-glycosylation system. Here, we describe how we prepared properly folded EGF repeats using two different bacterial expression vectors, generated recombinant glycosyltransferases, and performed in vitro O-glycosylation and subsequent product analysis by mass spectrometry. The methods described here are useful for investigating the enzymatic activities of mutated glycosyltransferases, revealing the structural basis of the O-glycosylation mechanism by co-crystallization of the glycosyltransferase-EGF repeat complexes, or identifying potential inhibitors of these glycosyltransferases.