Abeta42-to-Abeta40- and angiotensin-converting activities in different domains of angiotensin-converting enzyme

Abstract

Amyloid beta-protein 1-42 (Abeta42) is believed to play a causative role in the development of Alzheimer disease (AD), although it is a minor part of Abeta. In contrast, Abeta40 is the predominant secreted form of Abeta and recent studies have suggested that Abeta40 has neuroprotective effects and inhibits amyloid deposition. We have reported that angiotensin-converting enzyme (ACE) converts Abeta42 to Abeta40, and its inhibition enhances brain Abeta42 deposition (Zou, K., Yamaguchi, H., Akatsu, H., Sakamoto, T., Ko, M., Mizoguchi, K., Gong, J. S., Yu, W., Yamamoto, T., Kosaka, K., Yanagisawa, K., and Michikawa, M. (2007) J. Neurosci. 27, 8628-8635). ACE has two homologous domains, each having a functional active site. In the present study, we identified the domain of ACE, which is responsible for converting Abeta42 to Abeta40. Interestingly, Abeta42-to-Abeta40-converting activity is solely found in the N-domain of ACE and the angiotensin-converting activity is found predominantly in the C-domain of ACE. We also found that the N-linked glycosylation is essential for both Abeta42-to-Abeta40- and angiotensin-converting activities and that unglycosylated ACE rapidly degraded. The domain-specific converting activity of ACE suggests that ACE inhibitors could be designed to specifically target the angiotensin-converting C-domain, without inhibiting the Abeta42-to-Abeta40-converting activity of ACE or increasing neurotoxic Abeta42.

FIGURE 1.

Identification of N-domain-specific Aβ42-to-Aβ40-converting activity of ACE. A, schematic representation of the human ACE and recombinant ACE proteins. The wild-type ACE protein contains a signal peptide (SP), a single transmembrane domain (TM), and two homologous catalytic domains (light blue box). Recombinant ACE proteins, F-ACE, N-ACE, and C-ACE, contain 6 histidine residues (yellow box) at the C terminus and a signal peptide at the N terminus. B, COS7 cells transfected with empty vector or cells stably expressing F-ACE, N-ACE, or C-ACE were lysed in radioimmune precipitation assay buffer. Western blots of 20 μg of total protein from the cells or 2 μg of ACE isolated from the culture medium were probed with a polyclonal anti-ACE antibody. C, ACE activity was measured by incubating 0.5 μm F-ACE, N-ACE, or C-ACE with the substrate Hip-His-Leu for 15 min at 37 °C. N-ACE has markedly reduced ACE activity compared with C-ACE. Values represent the means + S.E.; n = 3; *, p < 0.001, Bonferroni/Dunn test. D, specificities of monoclonal anti-Aβ40 (1A10) and polyclonal anti-Aβ42 antibodies were confirmed by Western blot of 0.1 μg of Aβ40 and Aβ42. E, F-, N-, and C-ACE were mixed with synthetic Aβ42 and incubated at 37 °C for 0.5, 1, or 2 h. Western blots of the mixture were probed with anti-Aβ40 and anti-Aβ42 antibodies. In contrast to the ACE activity, the Aβ42-to-Aβ40-converting activity was solely detected in N-ACE. F, generation of Aβ40 and the degradation of Aβ42 were determined by densitometry.

FIGURE 2.

MALDI-TOF-MS analysis for Aβ42 degradation by F-ACE, N-ACE, or C-ACE. A, Aβ42 (80 μm) was incubated with 0.5 μm purified F-ACE, N-ACE, or C-ACE at 37 °C for 2 h, then captopril (10 μm) was added after incubation to stop the digestion. 1 μl of the mixture was subjected to MALDI-TOF-MS analysis. F-ACE and N-ACE generated Aβ1–40, whereas C-ACE did not. B, 1 μl of F-ACE, N-ACE, or C-ACE alone incubated at 37 °C for 2 h was subjected to MALDI-TOF-MS analysis, and a peptide signal was not detected.

FIGURE 3.

Site-directed mutated ACE proteins exhibit domain-specific Aβ42-to-Aβ40- and angiotensin-converting activity. A, schematic representation of human ACE and the mutant positions. The two ACE zinc metalloprotease active site glutamates (amino acids 362 in the N-domain and 960 in the C-domain) were changed to aspartates. B, fibroblasts were transiently transfected with empty vector, wtACE or mutant ACE plasmids and the expression of ACE proteins was detected by Western blotting using a polyclonal anti-ACE antibody. C, ACE activity was measured by incubating 5 μg of protein of cell lysate with the substrate Hip-His-Leu for 10 min at 37 °C. ACE activity in cell lysate was clearly detected in wtACE and E362D. C-domain inactive ACE protein, E960D, showed an extremely low ACE activity; and double mutants in both domains of ACE, E362/960D, did not show ACE activity. ACE activity was clearly inhibited by captopril (1 μm) treatment. D, ACE in cell lysate (4 mg of protein) from each transfected cell line was immunoprecipitated by 5 μg of polyclonal anti-ACE antibody and 100 μl of protein G-Sepharose. Immunoprecipitated ACE was then incubated with synthetic Aβ42 and the generation of Aβ40 was detected by Western blotting. SP, signal peptide; TM, transmembrane.

FIGURE 4.

Characterization of ACE glycosylation and role of the glycosylation in ACE activity and Aβ42-to-Aβ40-converting activity. A, 5 μg of purified human kidney ACE, F-ACE, N-ACE, and C-ACE were deglycosylated with 1 μl of PNGase F, O-glycanase, and/or sialidase A for 1 h at 37 °C. PNGase F alone was able to remove all glycosylation of ACE. B, ACE activity of PNGase F-deglycosylated human kidney ACE was measured immediately after deglycosylation using an ACE colorimetric kit. ACE activity was almost completely abolished by N-deglycosylation. C, 80 μl of human kidney ACE (0.5 μm) with or without N-deglycosylation was mixed with synthetic Aβ42 (40 μm) and incubated at 37 °C. 10 μl of the mixture were collected at various incubation time points and subjected to Western blot analysis. Deglycosylated ACE showed no Aβ42-to-Aβ40-converting activity, whereas the Aβ42-degrading activity remained. D, 40 μl of recombinant F-, N-, and C-ACE proteins (0.5 μm) were deglycosylated and mixed with Aβ42 and incubated at 37 °C for 1, 2, or 16 h. Aβ42-to-Aβ40-converting activity was not detected in either deglycosylated F-ACE or deglycosylated N-ACE, whereas all the deglycosylated ACE showed an Aβ42-degrading activity.