Click Chemistry-mediated Biotinylation Reveals a Function for the Protease BACE1 in Modulating the Neuronal Surface Glycoproteome

Julia Herber,Jasenka Njavro,Regina Feederle,Ute Schepers,Ulrike C Müller,Stefan Bräse,Stephan A Müller,Stefan F Lichtenthaler

doi:10.1074/mcp.ra118.000608

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, 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 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 cell surface proteins. Nearly 700 transmembrane glycoproteins were consistently identified at the surface of primary neurons. To demonstrate the utility of SUSPECS, we applied it to the protease BACE1, which is a key drug target in Alzheimer's disease. Pharmacological BACE1-inhibition selectively remodeled the neuronal surface glycoproteome, resulting in up to 7-fold increased abundance of the BACE1 substrates APP, APLP1, SEZ6, SEZ6L, CNTN2, and CHL1, whereas other substrates were not or only mildly affected. Interestingly, protein changes at the cell surface only partly correlated with changes in the secretome. Several altered proteins were validated by immunoblots in neurons and mouse brains. Apparent nonsubstrates, such as TSPAN6, were also increased, indicating that BACE1-inhibition may lead to unexpected secondary effects. In summary, SUSPECS is broadly useful for determination of the surface glycoproteome and its correlation with the secretome.

Highlights

The cell surface proteome is dynamic and has fundamental roles in cell signaling
To be able to compare secretome changes to alterations in the surface proteome using material from the same cellular experiment, we first set up a workflow referred to as ‘Surface-Spanning Protein Enrichment with Click Sugars’ (SUSPECS), where secretome protein enrichment with click sugars” (SPECS)-like labeling is used to assess the surface proteome of glycosylated membrane proteins (Fig. 1)
Cell surface membrane proteins have a fundamental role for cell signaling in health and disease and may be proteolytically released into a cell’s secretome, where they can have additional functions in cell-cell communication

Summary

EXPERIMENTAL PROCEDURES

The following antibodies were used: polyclonal DNER (R&D Systems, AF2254), monoclonal SEZ6 clone 14E5 [19] and SEZ6L (R&D Systems, MN; AF4804), SEZ6L2 (R&D Systems, AF4916), monoclonal BACE1 clone 3D5 (kindly provided by Robert Vassar), polyclonal TSPAN6 (Abgent, CA; AP9224b), monoclonal MMP17 clone EP1270Y (Abcam, Cambridge, UK; ab51075), monoclonal Golgin-97 (Invitrogen, CA; A21270), monoclonal NEO1 clone 21A8 [11], polyclonal RGMa (R&D Systems, AF2458), monoclonal GluR2 (MAB397, Millipore, Merck, Darmstadt, Germany), polyclonal Calnexin (Enzo, Stressgen, Farmingdale, NY, ADI-SPA-860), monoclonal ␤-actin clone AC-74 (Sigma, Merck; A5316), HRP coupled anti-mouse (Promega, Mannheim, Germany; W402B) and anti-rabbit secondary (Promega, W401B), HRP-coupled anti-goat (Santa Cruz, sc-2020), anti-rat (Santa Cruz, TX; sc2006) and anti-sheep (Santa Cruz, sc-2473). To remove sulfo-DBCOBiotin in excess, cells were washed twice with 1ϫ PBS and lysed in STET-lysis buffer (150 mM NaCl, 50 mM Tris (pH 7.5), 2 mM EDTA, 1% Triton X-100) with protease inhibitor. For relative quantification the LFQ intensity ratios of C3 and vehicle (DMSO) treated samples were calculated separately for each biological replicate (n ϭ 6) to account for experimental variations of different neuronal cell preparation batches. Pellets (resulting from the first centrifugation step) were washed with 1ϫ PBS to remove soluble proteins and lysed afterward in SDS buffer (50 mM Tris (pH 8), 150 mM NaCl, 2 mM EDTA, 1% SDS) with protease inhibitor. Immunoblot experiments were statistically tested by applying the Mann-Whitney-U test

RESULTS

Other categories

B SURFACE

DISCUSSION