Journal: The Journal of Biological Chemistry

Article Title: Metalloprotease Meprin ? Generates Nontoxic N-terminal Amyloid Precursor Protein Fragments in Vivo *

doi: 10.1074/jbc.M111.252718

Figure Lengend Snippet: a, processing of recombinant human APP isoforms by meprin β. APP wt, schematic structure of APP and alternative spliced variants is shown. Only the full-length APP770 isoform contains both the Kunitz protease inhibitor (KPI) and additionally the Ox2 domain; the latter is absent in APP751. AICD = APP intracellular domain. HisAPP, APP751 and -695 were C-terminally truncated at position 613 or 669, respectively, to generate soluble protein. The insertion of an N-terminal His6 tag (6xHis) allowed affinity purification of the recombinant enzymes by Ni-NTA chromatography. CuBD, copper-binding domain; E2, conserved region of the central APP domain; GFLD, N-terminal growth factor-like domain. b, purified proteins were analyzed by 10% SDS-PAGE and subsequently Coomassie staining (lanes 1–3 and 4–6) as follows: starting material (lanes 1 and 4), washing material (lanes 2 and 5), and the protein sample purified with 50 mm imidazole (lanes 3 and 6). Additionally, proteins were transferred to polyvinylidene fluoride membrane and probed with a monoclonal penta-His (histidine) antibody and a polyclonal antibody specific for the N terminus of APP (anti-N-APP), indicating molecular masses of 100 kDa for APP695 and 110 kDa for APP751. c, proteolytic processing of APP isoforms by meprin β. Recombinant APP770 and purified APP variants 751 and 695 were incubated with meprin β at 37 °C for 1, 5, and 15 min and 30, 60, and 120 min, respectively. After immunoblotting, membranes were exposed to different antibodies (22C11 and Penta His, supplemental Fig. S2, anti-N-APP) specific for certain APP regions as indicated in a. Smaller fragments of processed APP695 about 20 kDa (N-APP20, upper arrow) and 11 kDa (N-APP11, lower arrow) were further analyzed (c). The 11-kDa (N-APP11) fragment starts with the mature N terminus of APP retrieved by Edman degradation.

Article Snippet: Human APP770 (R & D Systems), APP751, and APP695 were incubated with meprin β for given periods of time at 37 °C in a 100:1 molar ratio.

Techniques: Recombinant, Protease Inhibitor, Affinity Purification, Chromatography, Binding Assay, Purification, SDS Page, Staining, Membrane, Incubation, Western Blot