O Linked N Acetylglucosamine Is Present on the Extracellular Domain of Notch Receptors*
Rare types of glycosylation often occur in a domain specific manner and are involved in specific biological processes. In particular, O fucose glycans are reported to regulate the functions of EGF domain containing proteins such as Notch receptors. In the course of mass spectrometric analysis of O glycans displayed on Drosophila Notch receptors expressed in S2 cells, we found an unusual O linked N acetylhexosamine (HexNAc) modification which occurs at a site distinct from those of O fucose and O glucose glycosylations. Modification site mapping by mass spectrometry and amino acid substitution studies revealed that O HexNAc modification occurs on a serine or threonine located between the fifth and sixth cysteines within the EGF domain. This modification occurs simultaneously along with other closely positioned O glycosylations. This modification was determined to be O b GlcNAc by galactosyltransferase labeling and b N acetyl hexosaminidase digestion experiments and by immunoblotting with a specific antibody. O GlcNAc modification occurs at multiple sites on Notch epidermal growth factor repeats. O GlcNAc modification was also found on the extracellular domain of Delta, a ligand for Notch receptors. Although the O GlcNAc modification is known to regulate a wide range of cellular processes, the list of known modified proteins has previously been limited to intracellular proteins in animals. Thus, the finding of O GlcNAc modification in extracellular environments predicts a distinct glycosylation process that might be associated with a novel regulatory mechanism for Notch receptor activity.
Journal of Biological Chemistry