Human membrane metallo-endopeptidase-like protein degrades both beta-amyloid 42 and beta-amyloid 40

Abstract

Beta-amyloid (Abeta) degrading endopeptidases are thought to protect against Alzheimer's disease (AD) and are potentially therapeutic. Of particular interest are endopeptidases that are blocked by thiorphan and phosphoramidon (T/P), as these inhibitors rapidly induce Abeta deposition in rodents. Neprilysin (NEP) is the best known target of T/P; however neprilysin knockout results in only modest Abeta increases insufficient to induce deposition. Therefore, other endopeptidases targeted by T/P must be critical for Abeta catabolism. Another candidate is the T/P sensitive membrane metallo-endopeptidase-like protein (MMEL), a close homolog of neprilysin. The endopeptidase properties of beta and gamma splice forms of human MMEL were determined in HEK293T cells transduced with the human cDNAs for the two splice forms; this showed degradation of both Abeta(42) and Abeta(40) by hMMEL-beta but not hMMEL-gamma. hMMEL-beta activity was found at the extracellular surface with no significant secreted activity. hMMEL-gamma was not expressed at the extracellular surface. Finally, it was found that hMMEL cleaves Abeta near the alpha-secretase site (producing Abeta(1-17)>>Abeta(1-16)). These data establish hMMEL as a mediator of Abeta catabolism and raise the possibility of its involvement in the etiology of AD and as a target for intervention.

Figure 1. Representations of the murine and human forms of MMEL

Alternate exons are shown in grey boxes. These are predicted type-II integral membrane proteins with short N-terminal cytoplasmic tails and a single transmembrane (TM) spanning region. The arrow indicates a consensus furin-like cleavage sequence. Murine MMEL-α has been shown to be a membrane bound protein which can degrade Aβ₄₀. Murine MMEL-β has been shown to be a secreted protein which lacks Aβ degrading activity. Human MMEL-β is the corresponding homolog of mMMEL-β. Human MMEL-γ does not contain a single alternate exon while hMMEL-δ utilizes an alternate splice acceptor site near its C-terminus resulting in a frameshift mutation / truncation lacking the critical zinc binding motif [HEITH].

Figure 2. The β splice form of MMEL can degrade Aβ

Aβ₄₂ (A, C) and Aβ₄₀ (B, D) were degraded by hMMEL-β but not hMMEL-γ in co-infection (A, B) and co-culture assays (C, D). Data are presented as the % Aβ remaining compared to a control condition (i.e. cells expressing GFP and APP). Methods: Lentiviral vectors were produced as previously described (Marr et al. 2003, Singer et al. 2005). Briefly, HEK293T cells were transfected (Ca-phosphate method) (Graham and van der Eb, 1973, Tiscornia et al., 2006) with vector and packaging plasmids and the cell culture supernatants containing virus collected. For co-infection, HEK293T cells were transduced with the lenti-APP vector (100 μl) and then split into wells of a 24 well plate 1 day later. After this the APP-infected wells were co-infected with 100 μl of a lenti-endopeptidase or control vector. 2 days later the culture medium was replaced with serum free medium (OptiMEM + 1% antibiotic/antimycotic, Gibco, Grand Island, NY) and the medium collected 24 hrs later for Aβ quantitation using specific ELISA (BioSource, Nivelles, Belgium). For co-culture experiments, cells were infected with the lenti-APP vector preparation (100μl). Also separate wells of cells were infected with a lenti-endopeptidase or control vector (100μl). The next day the APP-infected cells were split and co-cultured with endopeptidase or control infected cells. These cells were cultured for 2 days and Aβ-containing medium collected and assayed as done for the co-infection assay (above). Experiments using recombinant Aβ₄₂ and Aβ₄₀ established that the cross reactivity between Aβ isoforms on the specific Aβ ELISA kits were < 0.1% as reported by the manufacturer (not shown). The data are expressed as mean ±S.E.M. (n = 5, *p<0.05, **p<0.01).

Figure 3. Aβ_1-17 and Aβ_1-16 are preferred cleavage products produced by hMMEL-β

Incubation of synthetic Aβ with recombinant hMMEL-β produced major products corresponding to Aβ_1-17 and Aβ_1-16 (A). A representative MS profile is shown below (10 min incubation) (B). Methods: Aβ₄₀ or Aβ₄₂ (Quality Controlled Biochemicals, Hopkinton, MA) was dissolved in dimethyl sulfoxide at 20 mg/ml as a stock solution. 7 μg of Aβ was incubated with 1.7 μg of recombinant hNEP2 (MMEL) ∼100:1 [substrate:enzyme] (R&D Systems, Minneapolis, MN) in 50 μl of 150 mM NaCl, 50 mM Tris, pH 7.5 at 37° C. The reactions were stopped with 0.2% formate. Mass spectrometry analysis was performed on a Voyager DE-STR Biospectrometry workstation (Applied Biosystems, Foster City, CA; as described in Tullai et al., 2000) with a-cyano-4-hydroxycinnamic acid as matrix in delayed extraction and reflector mode with 200 shots after calibration.

Figure 4. hMMELβ is present at the cell surface

hMMEL-β and γ was associated with the cell lysate and hMMEL-β was also detected in the cell culture supernatant by immunoblot. Recombinant human MMEL-β (rhMMEL-ß, R&D Systems) was included as a positive control (5ng) (A). hMMEL-β but not γ was found at the cell surface as immunodetected by flow cytometry. The histogram shows the number of cells (y-axis) that were counted at the specific intensity of green fluorescence (x-axis) (B). Aβ degrading activity was not transferable in the medium for hMMEL-β and NEP; however a secreted mutant of NEP (secNEP) degraded Aβ (C). Methods: Western blot: Transfected cells were homogenized in RIPA buffer with protease inhibitors (Complete mini™, Roche, Indianapolis, IN). Cell lysates and cell culture supernatants were then analyzed by SDS-PAGE / immunoblot (1° antibody - polyclonal goat anti-human MMEL2 (AF2340, R&D Systems) (1:5000)) and visualized by HRP-conjugated 2°- antibody and chemiluminescence (Lumi-Light^PLUS, Roche). Flow cytometry: Transfected cells were blocked in PBS + 5% donor goat serum (Gemini Bio-Products, West Sacramento, CA) for 30 minutes (4°C). Cells were then immunostained with a monoclonal mouse anti-human MMEL2 1° antibody (1:100) (MAB2340, R&D Systems) (1 hr, 4°C), and goat anti-mouse-IgG Alexa Fluor 488 2° antibody (1:20) (Invitrogen) (30 min, 4°C) before analysis by flow cytometry. Secreted Aß degrading activity: Transfected cell culture medium was collected and then mixed (1:1) with Aβ₄₀ containing medium and incubated (6hrs) / analyzed by specific ELISA (as above). The data are averages ± S.E.M. (n=3, **p<0.01).