Detecting post-translational modification, O-GlcNAc, on Munc18a protein.

Abstract

O-GlcNAcylation, an abundant occurring post-translational modification (PTM), has been identified to play a major role in regulating proteins involved in many biological cellular functions. More recently, O-linked -N-acetylglucosamine (O-GlcNAc) has also been implicated to play important role in regulating brain function. Munc18a, an important protein that regulates the exocytosis of neurotransmitter, was identified to be O-GlcNAc modified at serine 511 using mass spectrometry. The aim of this study was to determine whether Munc18a could indeed be O-GlcNAc modified by using various biochemical analysis methods. Both wild-type and mutant Munc18a proteins were transfected into human embryonic kidney (HEK) 293 cells and targeted proteins were pulled-down and run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to detect O-GlcNAcylation. We observed that Munc18a can be O-GlcNAc modified by O-linked -N-acetylglucosamine transferase (OGT), but the specific O-GlcNAc sites responsible for this O-GlcNAcylation were not successfully identified. Further studies are necessary to identify O-GlcNAc sites of Munc18a, which would enable us to understand the physiological roles of post-translational modification on Munc18a.