O-GlcNAc modification of the extracellular domain of Notch receptors

Epidermal growth factor (EGF) domains are posttranslationally modified with unique O-linked glycans. The classical types of O-glycans on EGF domains are O-fucose and O-glucose glycans, found on many plasma glycoproteins and signaling molecules, whose biological functions have been demonstrated especially in the context of the Notch signaling pathway. We recently discovered O-GlcNAc modification as a new modification of the EGF domain that occurs on the conserved Ser/Thr residue located between the fifth and sixth cysteine residues within the EGF domain of Notch receptors in Drosophila. Here, we describe the methods employed to detect the O-GlcNAc modification of EGF repeats of Notch receptors. These methods include mass spectrometric analysis, galactosyltransferase labeling, immunoblotting with a specific antibody, and beta-N-acetyl-hexosaminidase digestion experiments. We also describe a method to detect O-GlcNAc transferase activity from crude membrane fraction proteins prepared from cultured S2 cells.