Surface glycoproteome of BACE1-inhibited neurons

ABSTRACT: The cell surface proteome is dynamic and has fundamental roles in cell signaling. Many surface membrane proteins are proteolytically released into a cell’s secretome, where they can have additional functions in cell-cell-communication. Yet, it remains challenging to determine the surface proteome and to compare it to the cell secretome, in particular under serum-containing cell culture conditions. Here, we set-up and evaluated the ‘surface-spanning protein enrichment with click sugars’ (SUSPECS) method for cell surface membrane glycoprotein biotinylation, enrichment and label-free quantitative mass spectrometry. SUSPECS is based on click chemistry-mediated labeling of glycoproteins, is fully compatible with labeling of living cells and can be combined with secretome analyses in the same experiment. Immunofluorescence-based confocal microscopy demonstrated that SUSPECS selectively labeled proteins at the cell surface, but not within cells. Nearly 700 transmembrane glycoproteins were quantified at the surface of primary murine neurons. To demonstrate the utility of SUSPECS, we tested how the protease BACE1, which is a key drug target in Alzheimer’s disease, affects the cell surface glycoproteome. Pharmacological inhibition of BACE1 selectively remodeled the neuronal surface proteome, resulting in up to seven-fold increased abundance of the BACE1 substrates APP, SEZ6, SEZ6L, CNTN2, CHL1 and L1, while other substrates were not or only mildly affected. Protein changes at the cell surface only partly correlated with changes in the secretome. Additionally, apparent non-substrates, such as TSPAN6, were also increased. Several altered proteins were validated by immunoblots in neurons and BACE1-deficient murine brains and indicate that BACE1-inhibition may lead to unexpected secondary effects.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Neuron Of Cerebral Cortex, Primary Cerebral Cortical Neuron�

SUBMITTER: Stephan Mueller  

LAB HEAD: Stefan Lichtenthaler

PROVIDER: PXD008619 | Pride | 2018-05-08

REPOSITORIES: Pride