Determination of the Proteolytic Cleavage Sites of the Amyloid Precursor-Like Protein 2 by the Proteases ADAM10, BACE1 and c-Secretase

Regulated intramembrane proteolysis of the amyloid precursor protein (APP) by the protease activities a-, band csecretase controls the generation of the neurotoxic amyloid b peptide. APLP2, the amyloid precursor-like protein 2, is a homolog of APP, which shows functional overlap with APP, but lacks an amyloid b 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 b-secretase BACE1 and additionally by an a-secretase activity. The two metalloproteases ADAM10 and ADAM17 have been suggested as candidate APLP2 a-secretases in cell lines. Here, we used RNA interference and found that ADAM10, but not ADAM17, is required for the constitutive a-secretase cleavage of APLP2 in HEK293 and SH-SY5Y cells. Likewise, in primary murine neurons knock-down of ADAM10 suppressed APLP2 a-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. c-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. Citation: Hogl S, Kuhn P-H, Colombo A, Lichtenthaler SF (2011) Determination of the Proteolytic Cleavage Sites of the Amyloid Precursor-Like Protein 2 by the Proteases ADAM10, BACE1 and c-Secretase. PLoS ONE 6(6): e21337. doi:10.1371/journal.pone.0021337 Editor: Koichi M. Iijima, Thomas Jefferson University, United States of America Received March 25, 2011; Accepted May 25, 2011; Published June 17, 2011 Copyright: 2011 Hogl et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: We thank the following institutions for financial support: the Deutsche Forschungsgemeinschaft for SFB596 project B12, the competence network degenerative dementias (BMBF) and the PPP program (DAAD) (all to SFL) and the Boehringer Ingelheim Foundation to SH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]