Constitutive and regulated alpha-secretase cleavage of Alzheimer's amyloid precursor protein by a disintegrin metalloprotease

Abstract

Amyloid beta peptide (Abeta), the principal proteinaceous component of amyloid plaques in brains of Alzheimer's disease patients, is derived by proteolytic cleavage of the amyloid precursor protein (APP). Proteolytic cleavage of APP by a putative alpha-secretase within the Abeta sequence precludes the formation of the amyloidogenic peptides and leads to the release of soluble APPsalpha into the medium. By overexpression of a disintegrin and metalloprotease (ADAM), classified as ADAM 10, in HEK 293 cells, basal and protein kinase C-stimulated alpha-secretase activity was increased severalfold. The proteolytically activated form of ADAM 10 was localized by cell surface biotinylation in the plasma membrane, but the majority of the proenzyme was found in the Golgi. These results support the view that APP is cleaved both at the cell surface and along the secretory pathway. Endogenous alpha-secretase activity was inhibited by a dominant negative form of ADAM 10 with a point mutation in the zinc binding site. Studies with purified ADAM 10 and Abeta fragments confirm the correct alpha-secretase cleavage site and demonstrate a dependence on the substrate's conformation. Our results provide evidence that ADAM 10 has alpha-secretase activity and many properties expected for the proteolytic processing of APP. Increases of its expression and activity might be beneficial for the treatment of Alzheimer's disease.

Figure 1

Cleavage of peptides spanning the α-secretase cleavage site of APP by ADAM 10. (A) Peptide substrates were incubated in cleavage buffer in the absence (Left) or in the presence of ADAM 10 (Right) for 6 hr at 37°C, followed by HPLC analysis. The asterisks indicate the peptide substrates and the numbers indicate the products generated as follows: 1, EVHHQK-OH; 2, LVFFAEDVGSNK-NH₂; 3, LVFFGEDVGSNK-NH₂. (B) CD spectra of APP peptides. Measurements were carried out in the presence of 0.5% SDS.

Figure 2

Expression and deglycosylation of ADAM 10 protease (A). After transfection of HEK and HEK APP₆₉₅ cells with ADAM 10 cDNA, cell lysates were immunoprecipitated with anti-HA antibody, subjected to deglycosylation with N-glycosidase F (PNGase F) (lane 3), or directly analyzed by 4–12% NuPAGE gel system and Western blot. (B) HEK and HEK ADAM 10 cells were surface-biotinylated. After immunoprecipitation with anti-HA antibody (16B12) and elution of the immune complexes, four-fifths of the eluate were incubated with immobilized streptavidin (lanes a), and the remaining one-fifth was directly applied to a 10% SDS/PAGE gel (lanes b) and then blotted onto PVDF membranes.

Figure 3

Colocalization of ADAM 10 and of a Golgi marker in HEK cells. Stably expressed ADAM 10 and the Golgi network were simultaneously immunostained. (Left) Anti-Golgi 58K protein. (Right) Anti-HA-epitope staining. The high degree of overlap of both immunoreactivities in the perinuclear region reveals a strong colocalization of ADAM 10 and the trans-Golgi marker. The scale bar is in μm.

Figure 4

Secretion of APPsα from HEK and HEK ADAM 10 cells. (A) Cells were incubated in the presence of indicated compound. After 4 hr, the medium was collected, and proteins were precipitated and subjected to immunoblot analysis with antibody 6E10 followed by a ³⁵S-labeled anti-mouse IgG antibody. (B) Quantitative analysis of secreted APPsα. The radioactive bands corresponding to APPsα were quantified with the Bio-Imaging analyzer model BAS-1800. The results are expressed as percentage of secreted APPsα in control HEK cells and are the averages ±SD of at least three experiments.

Figure 5

Effect of ADAM 10 on the production of APPsα and on the 10-kDa C-terminal fragment. (A) Cells were metabolically labeled with l-[³⁵S]methionine and [³⁵S]cysteine (200 μCi/ml) for 5 hr. Cell media were immunoprecipitated with antibody 1736 and analyzed by SDS/PAGE in 10% gels. (B) Cell lysates were immunoprecipitated with antibody C7. The samples were separated by 10–20% Tris/Tricine gel (Novex) and analyzed as described. (C) Quantitative analysis of holo-APP and p10. The values of p10 were normalized to the levels of holo-APP. The results are the averages ±SD of four experiments. Statistical significance between control cells and HEK ADAM 10 cells was determined by Student’s unpaired t test (∗, P < 0.005).

Figure 6

Inhibition effect of a dominant negative ADAM 10 protease form (DN). (A) Endogenous expression of ADAM 10 in HEK and MDBK cells detected by reverse transcriptase–PCR. In each case a 541-bp DNA fragment could be amplified. As control, RNA from HEK cells was treated with RNase A before reverse transcription (St, DNA molecular weight marker). (B) Quantitative analysis of secreted APPsα after immunoblot analysis. The results are expressed as percentage of secreted APPsα in control HEK cells and are the averages ±SD of at least three experiments. Statistical significance between control cells and HEK/DN cells treated or untreated with PMA was determined by Student’s unpaired t test (∗, P < 0.005; ∗∗, P < 0.001).