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Generation of aggregation prone N-terminally truncated amyloid β peptides by meprin β depends on the sequence specificity at the cleavage site.

Schönherr C, Bien J, Isbert S, Wichert R, Prox J, Altmeppen H, Kumar S, Walter J, Lichtenthaler SF, Weggen S, Glatzel M, Becker-Pauly C, Pietrzik CU - Mol Neurodegener (2016)

Bottom Line: We further analyzed the cellular interaction of APP and meprin β and found that cleavage of APP by meprin β occurs prior to endocytosis.The N-terminally truncated Aβ2-40 variant shows increased aggregation propensity compared to Aβ1-40 and acts even as a seed for Aβ1-40 aggregation.Concluding, we propose that meprin β may be involved in the generation of N-terminally truncated Aβ2-x peptides of APP, but acts independently from BACE-1.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathobiochemistry, University Medical Center of the Johannes Gutenberg University of Mainz, Duesbergweg 6, 55128, Mainz, Germany.

ABSTRACT

Background: The metalloprotease meprin β cleaves the Alzheimer's Disease (AD) relevant amyloid precursor protein (APP) as a β-secretase reminiscent of BACE-1, however, predominantly generating N-terminally truncated Aβ2-x variants.

Results: Herein, we observed increased endogenous sAPPα levels in the brains of meprin β knock-out (ko) mice compared to wild-type controls. We further analyzed the cellular interaction of APP and meprin β and found that cleavage of APP by meprin β occurs prior to endocytosis. The N-terminally truncated Aβ2-40 variant shows increased aggregation propensity compared to Aβ1-40 and acts even as a seed for Aβ1-40 aggregation. Additionally, we observed that different APP mutants affect the catalytic properties of meprin β and that, interestingly, meprin β is unable to generate N-terminally truncated Aβ peptides from Swedish mutant APP (APPswe).

Conclusion: Concluding, we propose that meprin β may be involved in the generation of N-terminally truncated Aβ2-x peptides of APP, but acts independently from BACE-1.

No MeSH data available.


Related in: MedlinePlus

The “protective” APP A673T mutation decreases cleavage by meprin β. a, b 15 nM recombinant meprin β was incubated with synthetic APP peptides at 37 °C. HPLC analysis showed that processing kinetics of APP A673T were decreased (b) compared to wt APP (a) (see also Additional file 4). c Supernatants of HEK-293 T cells, transiently transfected with APPwt or APP A673T mutant and co-transfected with meprin β or empty vector were immunoprecipitated with anti-Aβ 6E10-Dynabeads, subsequently separated on an 8 M urea gel and probed with 6E10. The Aβ2-40 band, visible in samples transfected with APPwt and meprin β, is slightly shifted in samples transfected with APP A673T and meprin β. All samples were run on one gel but rearranged for better presentation. d A significant decrease of the Aβ2-40/1-40 ratio was observed in culture supernatants of cells co-transfected with APP A673T and meprin β compared to cells co-transfected with APPwt and meprin β (graph shows mean ± SEM (n = 5); statistical significance: *, p = 0.0317; t-test)
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Fig6: The “protective” APP A673T mutation decreases cleavage by meprin β. a, b 15 nM recombinant meprin β was incubated with synthetic APP peptides at 37 °C. HPLC analysis showed that processing kinetics of APP A673T were decreased (b) compared to wt APP (a) (see also Additional file 4). c Supernatants of HEK-293 T cells, transiently transfected with APPwt or APP A673T mutant and co-transfected with meprin β or empty vector were immunoprecipitated with anti-Aβ 6E10-Dynabeads, subsequently separated on an 8 M urea gel and probed with 6E10. The Aβ2-40 band, visible in samples transfected with APPwt and meprin β, is slightly shifted in samples transfected with APP A673T and meprin β. All samples were run on one gel but rearranged for better presentation. d A significant decrease of the Aβ2-40/1-40 ratio was observed in culture supernatants of cells co-transfected with APP A673T and meprin β compared to cells co-transfected with APPwt and meprin β (graph shows mean ± SEM (n = 5); statistical significance: *, p = 0.0317; t-test)

Mentions: A recently described APP mutation in position 673 (A673T) has been shown to protect against AD as well as against cognitive decline in the elderly independently of AD [28–30]. This mutation is located adjacent to the β-secretase cleavage site in the Aβ sequence at p2 and reduces Aβ generation by 40 % in vitro. [28]. According to the findings reported above we speculated that this amino acid exchange (A673T) may also influence the affinity of meprin β towards APP. To investigate the influence of this mutation on meprin β cleavage of APP, we performed a cleavage assay using recombinant enzyme and synthetic peptides including the A673T mutation. HPLC and subsequent MALDI analysis revealed preferred cleavage of the wt over the A673T APP peptide by meprin β (Fig. 6a, b; Additional file 4). Indeed, meprin β prefers alanine over threonine in P1’ position [24], which may explain reduced cleavage of APP A673T by meprin β.Fig. 6


Generation of aggregation prone N-terminally truncated amyloid β peptides by meprin β depends on the sequence specificity at the cleavage site.

Schönherr C, Bien J, Isbert S, Wichert R, Prox J, Altmeppen H, Kumar S, Walter J, Lichtenthaler SF, Weggen S, Glatzel M, Becker-Pauly C, Pietrzik CU - Mol Neurodegener (2016)

The “protective” APP A673T mutation decreases cleavage by meprin β. a, b 15 nM recombinant meprin β was incubated with synthetic APP peptides at 37 °C. HPLC analysis showed that processing kinetics of APP A673T were decreased (b) compared to wt APP (a) (see also Additional file 4). c Supernatants of HEK-293 T cells, transiently transfected with APPwt or APP A673T mutant and co-transfected with meprin β or empty vector were immunoprecipitated with anti-Aβ 6E10-Dynabeads, subsequently separated on an 8 M urea gel and probed with 6E10. The Aβ2-40 band, visible in samples transfected with APPwt and meprin β, is slightly shifted in samples transfected with APP A673T and meprin β. All samples were run on one gel but rearranged for better presentation. d A significant decrease of the Aβ2-40/1-40 ratio was observed in culture supernatants of cells co-transfected with APP A673T and meprin β compared to cells co-transfected with APPwt and meprin β (graph shows mean ± SEM (n = 5); statistical significance: *, p = 0.0317; t-test)
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Fig6: The “protective” APP A673T mutation decreases cleavage by meprin β. a, b 15 nM recombinant meprin β was incubated with synthetic APP peptides at 37 °C. HPLC analysis showed that processing kinetics of APP A673T were decreased (b) compared to wt APP (a) (see also Additional file 4). c Supernatants of HEK-293 T cells, transiently transfected with APPwt or APP A673T mutant and co-transfected with meprin β or empty vector were immunoprecipitated with anti-Aβ 6E10-Dynabeads, subsequently separated on an 8 M urea gel and probed with 6E10. The Aβ2-40 band, visible in samples transfected with APPwt and meprin β, is slightly shifted in samples transfected with APP A673T and meprin β. All samples were run on one gel but rearranged for better presentation. d A significant decrease of the Aβ2-40/1-40 ratio was observed in culture supernatants of cells co-transfected with APP A673T and meprin β compared to cells co-transfected with APPwt and meprin β (graph shows mean ± SEM (n = 5); statistical significance: *, p = 0.0317; t-test)
Mentions: A recently described APP mutation in position 673 (A673T) has been shown to protect against AD as well as against cognitive decline in the elderly independently of AD [28–30]. This mutation is located adjacent to the β-secretase cleavage site in the Aβ sequence at p2 and reduces Aβ generation by 40 % in vitro. [28]. According to the findings reported above we speculated that this amino acid exchange (A673T) may also influence the affinity of meprin β towards APP. To investigate the influence of this mutation on meprin β cleavage of APP, we performed a cleavage assay using recombinant enzyme and synthetic peptides including the A673T mutation. HPLC and subsequent MALDI analysis revealed preferred cleavage of the wt over the A673T APP peptide by meprin β (Fig. 6a, b; Additional file 4). Indeed, meprin β prefers alanine over threonine in P1’ position [24], which may explain reduced cleavage of APP A673T by meprin β.Fig. 6

Bottom Line: We further analyzed the cellular interaction of APP and meprin β and found that cleavage of APP by meprin β occurs prior to endocytosis.The N-terminally truncated Aβ2-40 variant shows increased aggregation propensity compared to Aβ1-40 and acts even as a seed for Aβ1-40 aggregation.Concluding, we propose that meprin β may be involved in the generation of N-terminally truncated Aβ2-x peptides of APP, but acts independently from BACE-1.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathobiochemistry, University Medical Center of the Johannes Gutenberg University of Mainz, Duesbergweg 6, 55128, Mainz, Germany.

ABSTRACT

Background: The metalloprotease meprin β cleaves the Alzheimer's Disease (AD) relevant amyloid precursor protein (APP) as a β-secretase reminiscent of BACE-1, however, predominantly generating N-terminally truncated Aβ2-x variants.

Results: Herein, we observed increased endogenous sAPPα levels in the brains of meprin β knock-out (ko) mice compared to wild-type controls. We further analyzed the cellular interaction of APP and meprin β and found that cleavage of APP by meprin β occurs prior to endocytosis. The N-terminally truncated Aβ2-40 variant shows increased aggregation propensity compared to Aβ1-40 and acts even as a seed for Aβ1-40 aggregation. Additionally, we observed that different APP mutants affect the catalytic properties of meprin β and that, interestingly, meprin β is unable to generate N-terminally truncated Aβ peptides from Swedish mutant APP (APPswe).

Conclusion: Concluding, we propose that meprin β may be involved in the generation of N-terminally truncated Aβ2-x peptides of APP, but acts independently from BACE-1.

No MeSH data available.


Related in: MedlinePlus