Determination of the proteolytic cleavage sites of the amyloid precursor-like protein 2 by the proteases ADAM10, BACE1 and γ-secretase.

Regulated intramembrane proteolysis of the amyloid precursor
protein (APP) by the protease activities α-, β- and γ-secretase
controls the generation of the neurotoxic amyloid β peptide. APLP2,
the amyloid precursor-like protein 2, is a homolog of APP, which
shows functional overlap with APP, but lacks an amyloid β domain.
Compared to APP, less is known about the proteolytic processing of
APLP2, in particular in neurons, and the cleavage sites have not
yet been determined. APLP2 is cleaved by the β-secretase BACE1 and
additionally by an α-secretase activity. The two metalloproteases
ADAM10 and ADAM17 have been suggested as candidate APLP2
α-secretases in cell lines. Here, we used RNA interference and
found that ADAM10, but not ADAM17, is required for the constitutive
α-secretase cleavage of APLP2 in HEK293 and SH-SY5Y cells.
Likewise, in primary murine neurons knock-down of ADAM10 suppressed
APLP2 α-secretase cleavage. Using mass spectrometry we determined
the proteolytic cleavage sites in the APLP2 sequence. ADAM10 was
found to cleave APLP2 after arginine 670, whereas BACE1 cleaves
after leucine 659. Both cleavage sites are located in close
proximity to the membrane. γ-secretase cleavage was found to occur
at different peptide bonds between alanine 694 and valine 700,
which is close to the N-terminus of the predicted APLP2
transmembrane domain. Determination of the APLP2 cleavage sites
enables functional studies of the different APLP2 ectodomain
fragments and the production of cleavage-site specific antibodies
for APLP2, which may be used for biomarker development.