title: Determination of the proteolytic cleavage sites of the amyloid precursor-like protein 2 by the proteases ADAM10, BACE1 and γ-secretase.
abstract: 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.
authors.affiliation: German Center for Neurodegenerative Diseases, Munich, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Hogl
authors.forename: Peer-Hendrik
authors.initials: PH
authors.lastname: Kuhn
authors.forename: Alessio
authors.initials: A
authors.lastname: Colombo
authors.forename: Stefan F
authors.initials: SF
authors.lastname: Lichtenthaler
mesh_terms: D051722:ADAM Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000072197:ADAM10 Protein; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001120:Arginine; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D001665:Binding Sites; D045744:Cell Line, Tumor; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D011485:Protein Binding; D014633:Valine
chemical_list: C084104:APLP2 protein, human; D016564:Amyloid beta-Protein Precursor; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D001120:Arginine; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; D051722:ADAM Proteins; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human; D014633:Valine