Identification of β-Secretase (BACE1) Substrates Using Quantitative Proteomics
Figure 3
BACE1 shedding of GPI-linked and type II transmembrane proteins.
Identified BACE1 substrates ephrin-A5 and GOLIM4 were cloned, FLAG-tagged, and stably expressed in HEK cells that express BACE1 or empty vector as control. The left column shows Western blots of cell lysates, and the right column shows blots of conditioned medium. Cells were treated with the β-secretase inhibitor C3 to confirm the necessity of BACE1 activity for ectodomain shedding. (A) Ephrin-A5, a GPI-linked protein, was robustly expressed and produced two prominent bands, the lower presumably representing the processed and mature GPI-linked form. BACE1 activity decreased the levels of full-length protein, and the shed product was visible within the cellular lysate (left panel). Conditioned medium revealed one minor (ephrin-A5sα) and one major (ephrin-A5sβ) band indicative of shed ephrin-A5, the major band corresponding to the BACE1 cleavage product (right panel). (B) GOLIM4, a type II transmembrane protein, was poorly expressed in cellular lysates (left panel), but accumulation of the shed ectodomain was found in conditioned medium of BACE1 expressing cells (right panel).