Identification of ceramide binding proteins in neuronal cells: a critical point of view

Abstract

Much discussion has centered on the biochemical mechanism by which ceramide is produced and functions as a signalling molecule in cells. To identify proteins involved in ceramide signalling, we synthesized a radioactively labelled ceramide analogue equipped with a photosensitive group: N-(p-trifluoromethyl-diazirinyl)phenyl-ethyl-2-[35S]-2-thioacetyl-D-erythro-C18-sphingosine ([35S]-TDS-ceramide). This compound was then employed in photo-affinity labelling experiments in primary cultured cerebellar neurons. Due to the hydrophobic nature of the compound, most of the cell-associated radioactivity was recovered in the lipid fraction while only about 0.1% of radioactivity was photocoupled to proteins. In order to improve protein labelling the cytosolic fraction of rapidly growing human neuroblastoma cells (SH-SY5Y) was isolated and subjected to ceramide affinity chromatography prior to photo-affinity labelling. Following electrophoresis proteins photocoupled to ceramide were identified by MALDI mass spectrometry in combination with tryptic digestion and turned out to be either cytoskeletal or stress proteins that are highly abundant in cytosol and contain at least one hydrophobic domain.