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Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein.

ABSTRACT: The cDNAs of two new human membrane-associated aspartic proteases, memapsin 1 and memapsin 2, have been cloned and sequenced. The deduced amino acid sequences show that each contains the typical pre, pro, and aspartic protease regions, but each also has a C-terminal extension of over 80 residues, which includes a single transmembrane domain and a C-terminal cytosolic domain. Memapsin 2 mRNA is abundant in human brain. The protease domain of memapsin 2 cDNA was expressed in Escherichia coli and was purified. Recombinant memapsin 2 specifically hydrolyzed peptides derived from the beta-secretase site of both the wild-type and Swedish mutant beta-amyloid precursor protein (APP) with over 60-fold increase of catalytic efficiency for the latter. Expression of APP and memapsin 2 in HeLa cells showed that memapsin 2 cleaved the beta-secretase site of APP intracellularly. These and other results suggest that memapsin 2 fits all of the criteria of beta-secretase, which catalyzes the rate-limiting step of the in vivo production of the beta-amyloid (Abeta) peptide leading to the progression of Alzheimer's disease. Recombinant memapsin 2 also cleaved a peptide derived from the processing site of presenilin 1, albeit with poor kinetic efficiency. Alignment of cleavage site sequences of peptides indicates that the specificity of memapsin 2 resides mainly at the S(1)' subsite, which prefers small side chains such as Ala, Ser, and Asp.

SUBMITTER: Lin X

PROVIDER: S-EPMC26455 | biostudies-literature | 2000 Feb

REPOSITORIES: biostudies-literature

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Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein.

Lin X X Koelsch G G Wu S S Downs D D Dashti A A Tang J J

Proceedings of the National Academy of Sciences of the United States of America 20000201 4

The cDNAs of two new human membrane-associated aspartic proteases, memapsin 1 and memapsin 2, have been cloned and sequenced. The deduced amino acid sequences show that each contains the typical pre, pro, and aspartic protease regions, but each also has a C-terminal extension of over 80 residues, which includes a single transmembrane domain and a C-terminal cytosolic domain. Memapsin 2 mRNA is abundant in human brain. The protease domain of memapsin 2 cDNA was expressed in Escherichia coli and w ...[more]

PMID: 10677483

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Project description:BackgroundThree amino acid differences between rodent and human APP affect medically important features, including β-secretase cleavage of APP and Aβ peptide aggregation (De Strooper et al., EMBO J 14:4932-38, 1995; Ueno et al., Biochemistry 53:7523-30, 2014; Bush, 2003, Trends Neurosci 26:207-14). Most rodent models for Alzheimer's disease (AD) are, therefore, based on the human APP sequence, expressed from artificial mini-genes randomly inserted in the rodent genome. While these models mimic rather well various biochemical aspects of the disease, such as Aβ-aggregation, they are also prone to overexpression artifacts and to complex phenotypical alterations, due to genes affected in or close to the insertion site(s) of the mini-genes (Sasaguri et al., EMBO J 36:2473-87, 2017; Goodwin et al., Genome Res 29:494-505, 2019). Knock-in strategies which introduce clinical mutants in a humanized endogenous rodent APP sequence (Saito et al., Nat Neurosci 17:661-3, 2014) represent useful improvements, but need to be compared with appropriate humanized wildtype (WT) mice.MethodsComputational modelling of the human β-CTF bound to BACE1 was used to study the differential processing of rodent and human APP. We humanized the three pivotal residues we identified G676R, F681Y and R684H (labeled according to the human APP770 isoform) in the mouse and rat genomes using a CRISPR-Cas9 approach. These new models, termed mouse and rat Apphu/hu, express APP from the endogenous promotor. We also introduced the early-onset familial Alzheimer's disease (FAD) mutation M139T into the endogenous Rat Psen1 gene.ResultsWe show that introducing these three amino acid substitutions into the rodent sequence lowers the affinity of the APP substrate for BACE1 cleavage. The effect on β-secretase processing was confirmed as both humanized rodent models produce three times more (human) Aβ compared to the original WT strain. These models represent suitable controls, or starting points, for studying the effect of transgenes or knock-in mutations on APP processing (Saito et al., Nat Neurosci 17:661-3, 2014). We introduced the early-onset familial Alzheimer's disease (FAD) mutation M139T into the endogenous Rat Psen1 gene and provide an initial characterization of Aβ processing in this novel rat AD model.ConclusionThe different humanized APP models (rat and mouse) expressing human Aβ and PSEN1 M139T are valuable controls to study APP processing in vivo allowing the use of a human Aβ ELISA which is more sensitive than the equivalent system for rodents. These animals will be made available to the research community.

Dataset Information

Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein.

Publications

Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein.

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