The metalloprotease meprin β generates amino terminal-truncated amyloid β peptide species

Abstract

The amyloid β (Aβ) peptide, which is abundantly found in the brains of patients suffering from Alzheimer disease, is central in the pathogenesis of this disease. Therefore, to understand the processing of the amyloid precursor protein (APP) is of critical importance. Recently, we demonstrated that the metalloprotease meprin β cleaves APP and liberates soluble N-terminal APP (N-APP) fragments. In this work, we present evidence that meprin β can also process APP in a manner reminiscent of β-secretase. We identified cleavage sites of meprin β in the amyloid β sequence of the wild type and Swedish mutant of APP at positions p1 and p2, thereby generating Aβ variants starting at the first or second amino acid residue. We observed even higher kinetic values for meprin β than BACE1 for both the wild type and the Swedish mutant APP form. This enzymatic activity of meprin β on APP and Aβ generation was also observed in the absence of BACE1/2 activity using a β-secretase inhibitor and BACE knock-out cells, indicating that meprin β acts independently of β-secretase.

FIGURE 1.

Catalytic properties and cleavage sites of meprin β within different APP peptides. A, the amino acid sequence of APP with the wild type Aβ region (highlighted in a gray box) is presented, indicating the α-, β-, and γ-secretase cleavage sites. The peptide sequences analyzed are displayed in letters in boldface type. Black arrows indicate the cleavage sites analyzed by MALDI-TOF. Sequences are shown in the one letter code. Sw, Swedish mutant. B, kinetic parameters of Aβ cleavage by meprin β. The calculated kinetic constants for the wt β-site fluorogenic substrate (Abz-VKMDAE-EDDnp) are displayed in the left panel, for the Swedish mutated β site (Abz-VNLDAE-EDDnp) on the right. Abz, aminobenzoic acid; EDDnp, ethylenediamine 2.4-dinitrophenyl; M, molar mass; v, velocity. C, fluorogenic APP substrates for kinetic calculations. The enzyme concentration used in all assays was 1 × 10⁻⁹ m, and substrate concentrations varied as indicated in B. Std., standard.

FIGURE 2.

Meprin β mRNA levels are increased in brains of AD patients compared with age-matched control individuals. Quantitative real-time PCR of total RNA extracted from 10 age-matched healthy individuals and 10 AD patients. Statistical analysis revealed significantly higher levels of meprin β mRNA in the brain samples obtained from AD patients. To compare levels of meprin β RNA, ΔC_T, mean values were analyzed. Statistical analysis was performed using the Mann-Whitney test (non-parametric, one-tailed t test, statistical significance, *, p < 0.05).

FIGURE 3.

Overexpression of meprin β and APP751 wt in HEK293T cells leads to an increase in Aβ production. HEK293T cells transiently overexpressing human APP751 wt isoform and/or human meprin β were treated with 100 μm actinonin, a meprin β (Mepβ) inhibitor; 5 μm DAPT, a γ-secretase inhibitor; 1 μm BACE inhibitor IV (BACE Inh), a β-secretase inhibitor, or DMSO as a vehicle control, overnight. The amount of secreted Aβ was detected in cell culture medium by immunoprecipitation/Western blotting with the IC-16 antibody. A, protein levels of APP, meprin β, and tubulin were detected in the corresponding cell lysates. B, diagram showing Aβ production, depicted as percentage of APP751 wt single transfected cells. Comparison between APP751 wt overexpressing and APP751 wt and meprin β co-expressing cells revealed a significant increase in Aβ secretion due to meprin β activity. The Aβ production was decreased after treatment with the γ-secretase inhibitor DAPT, suggesting that meprin β produced Aβ is dependent on γ-secretase activity. Treatment with the meprin β inhibitor actinonin but not with a β-secretase inhibitor decreased Aβ levels, demonstrating that the increased Aβ generation in APP and meprin β co-expressing cells was dependent on meprin β but not BACE1 activity (graph shows mean ± S.E. (n = 5); statistical significance: *, p < 0.05; **, p < 0.01; one-way analysis of variance).

FIGURE 4.

Actinonin has no effect on γ-secretase activity. HEK293T cells stably expressing APP695, which is C-terminally fused to the DNA binding domain Gal4, were treated with 100 μm actinonin or 5 μm DAPT for 2 h. AICD generation was detected in cell lysate using an anti-APP antibody, 396, and compared with untreated cells. Due to Gal4, the AICDs are stabilized, and signal detection is improved. After DAPT treatment, AICD generation was abolished, and the amount of CTFs fused to Gal4 but also endogenous CTF levels were increased. After treatment with actinonin, no difference in AICD generation could be observed, and CTF levels were comparable with untreated cells. Therefore, actinonin seems to have no effect on γ-secretase activity.

FIGURE 5.

Meprin β inhibition leads to a decrease in Aβ secretion. HEK293T cells transiently overexpressing APP751 wt were treated with 10 μm actinonin, a meprin β inhibitor, for 24 h in serum-free medium. A, total secreted Aβ was detected in the samples of cell culture medium by immunoprecipitation using the IC-16 antibody. Protein levels of carboxyl-terminal APP fragments, CTFs, mature (mAPP), immature APP (immAPP), and tubulin were detected in the samples of cell lysates using an anti-APP antibody, 369, and an anti-tubulin antibody, respectively. B, the Aβ signals, obtained with the IC-16 antibody, were quantified and depicted as percentage of vehicle control. Actinonin treatment caused a statistically significant reduction in Aβ secretion (graph shows mean ± S.E. (n = 5); ***, p = 0.0008, t test).

FIGURE 6.

MALDI-MS spectra of secreted Aβ from APP751 wt and meprin β single and co-expressing cells. HEK293T cells were co-transfected with APP751 wt and meprin β and single-transfected as control. Additionally, co-expressing cells were treated with 5 μm DAPT, or DMSO overnight. With the monoclonal antibody 4G8, immunoprecipitated Aβ was analyzed using MALDI MS analysis. Shown are the mass spectra of one representative measurement of three independent experiments. A, mass spectrum showing Aβ1–40 peptides detected in the medium of APP751 wt single-transfected cells. B, in meprin β single transfected cells, no Aβ signal could be detected. C, after APP and meprin β co-transfection, in addition to the Aβ1–40 signal, an amino-terminal truncated Aβ2–40 peptide could be observed. D, both Aβ1–40 and Aβ2–40 production could be abolished using DAPT, a γ-secretase inhibitor. E, DMSO did not influence the production of both Aβ species. In another approach, PS70 cells stably expressing meprin β (or GFP as a control) were additionally infected with an APP cDNA containing adenovirus. Additionally, cells were treated with 5 μm DAPT (H), 1 μm BACE inhibitor IV (I), 100 μm actinonin (J), or DMSO (F and G) as a vehicle control overnight. With the monoclonal antibody W0–2, immunoprecipitated Aβ was analyzed using MALDI MS analysis. Shown are the mass spectra of Aβ peptides of the different treated cells. F, in cells expressing APP and GFP, the mass spectrum shows Aβ1–40, Aβ1–39, representing a normal Aβ pattern. G, in cells expressing APP and meprin β, the Aβ1–40 peak increased and an additional peak representing Aβ2–40 appears due to meprin β cleavage. H, after DAPT treatment of cells co-expressing APP and meprin β, no Aβ signal could be detected, suggesting that also meprin β cleaved Aβ is dependent on γ-secretase cleavage. I, treatment of these cells with actinonin, an inhibitor for meprin β, resulted in a loss of the Aβ2–40 species, proofing the specificity of meprin β toward the Aβ sequence. J, treatment with BACE inhibitor IV did not abolish the Aβ1–40 or the Aβ2–40 signal (n = 1). Theoretical and observed masses for Aβ1–40 and Aβ2–40 are 4328.2/4328.3 and 4213.1/4213.3 Da, respectively. Aβ2–40 was corroborated by LIFT sequencing.

FIGURE 7.

Production of Aβ in BACE1/2 KO cells stably co-expressing the APP695 wt isoform and meprin β. BACE1/2 knock-out MEFs stably co-expressing APP695 wt and meprin β were generated. Where indicated, the cells were treated with 10 μm actinonin, a meprin β inhibitor; 5 μm DAPT, a γ-secretase inhibitor or with DMSO as a vector control overnight. A, total secreted Aβ was detected in the samples of cell culture medium by immunoprecipitation with the IC-16 antibody. As an Aβ standard peptide, a synthetic Aβ1–40 peptide was used (Genosphere Biotechnologies). All samples were analyzed on the same Western blot but in a different order and rearranged for better understanding. B, the Aβ signals, obtained with the IC-16 antibody, were quantified and depicted as percentage of APP695 wt single-expressing cells (graph shows mean ± S.E. (n = 4); statistical significance: *, p < 0.05; t test). C, protein levels of carboxyl-terminal APP fragments, CTFs, APP, and tubulin were detected in the samples of cell lysates. Meprin β expression was analyzed using the MEP1B antibody. In comparison with cells stably overexpressing APP695 wt alone, cells stably co-expressing APP695 wt and meprin β showed a significant increase in Aβ secretion due to meprin β activity even in the absence of β-secretases due to a knock-out of BACE1 and BACE2. The Aβ production could be decreased using DAPT, a γ-secretase inhibitor, again suggesting that also meprin β cleaved Aβ is dependent on γ-secretase activity.

FIGURE 8.

Exogenous meprin β generates N-APP20 fragments, whereas it has no effect on Aβ. Treatment of APP-overexpressing cells with different concentrations of exogenously added inactive (E90A) and active meprin β (Mepβ) was carried out for 5 h in serum-free medium. Cell culture medium was used to detect total, sAPP, and N-APP20 fragments with anti-N-APP antibody, 22C11 (anti-N-APP Ab). Total extracellular Aβ was detected in the samples of cell culture medium with IC-16 antibody, raised against a peptide containing the first 16 amino acids of the Aβ sequence. As a control, medium was treated with HEPES buffer only. Anti-N-APP antibody, 22C11, was used to detect total sAPP and N-APP20 fragments, resulting from sAPP cleavage carried out by the active meprin β. Only total sAPP was detected following the incubation of cell culture medium with the inactive meprin β.