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Herber, J.* ; Njavro, J.* ; Feederle, R. ; Schepers, U.* ; Müller, U.C.* ; Bräse, S.* ; Müller, S.A.* ; Lichtenthaler, S.F.*

Click chemistry-mediated biotinylation reveals a function for the protease BACE1 in modulating the neuronal surface glycoproteome.

Mol. Cell. Proteomics 17, 1487-1501 (2018)
Publ. Version/Full Text Postprint DOI
Open Access Green
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.
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Publication type Article: Journal article
Document type Scientific Article
Keywords Mass Spectrometry ; Alzheimer's Disease ; Cell Surface Display Technologies ; Click Chemistry ; Label-free Quantification ; Bace1 ; Cell Surface ; N-glycosylation; Amyloid Precursor Protein; Regulated Intramembrane Proteolysis; Beta-secretase Cleavage; Cleaving Enzyme; Proteomic Analysis; Gene-function; In-vivo; A-beta; Cells; Molecule
ISSN (print) / ISBN 1535-9476
e-ISSN 1535-9484
Quellenangaben Volume: 17, Issue: 8, Pages: 1487-1501 Article Number: , Supplement: ,
Publisher American Society for Biochemistry and Molecular Biology
Publishing Place 9650 Rockville Pike, Bethesda, Md 20814-3996 Usa
Reviewing status Peer reviewed
Institute(s) Monoclonal Antibody (IDO-MAB)