Biochemical and kinetic characterization of BACE1: investigation into the putative species‐specificity for β‐ and β′‐cleavage sites by human and murine BACE1

Abstract

b-amyloid peptides (Ab) are produced by a sequential cleavage of amyloid precursor protein (APP) by b-and c-secretases. The lack of Ab production in beta-APP cleaving enzyme (BACE1) -/mice suggests that BACE1 is the principal b-secretase in mammalian neurons. Transfection of human APP and BACE1 into neurons derived from wild-type and BACE1 -/mice supports cleavage of APP at the canonical b-secretase site. However, these studies also revealed an alternative BACE1 cleavage site in APP, designated as b¢, resulting in Ab peptides starting at Glu11. The apparent inability of human BACE1 to make this b¢-cleavage in murine APP, and vice versa, led to the hypothesis that this alternative cleavage was species-specific. In contrast, the results from human BACE1 transgenic mice demonstrated that the human BACE1 is able to cleave the endogenous murine APP at the b¢-cleavage site. To address this discrepancy, we designed fluorescent resonance energy transfer peptide substrates containing the b-and b¢-cleavage sites within human and murine APP to compare: (i) the enzymatic efficiency; (ii) binding kinetics of a BACE1 active site inhibitor LY2039911; and (iii) the pharmacological profiles for human and murine recombinant BACE1. Both BACE1 orthologs were able to cleave APP at the b-and b¢-sites, although with different efficiencies. Moreover, the inhibitory potency of LY2039911 toward recombinant human and native BACE1 from mouse or guinea pig was indistinguishable. In summary, we have demonstrated, for the first time, that recombinant BACE1 can recognize and cleave APP peptide substrates at the postulated b¢-cleavage site. It does not appear to be a significant species specificity to this cleavage. Keywords: amyloid precursor protein, b-amyloid precursor protein cleaving enzyme, b-cleavage, b¢-cleavage, speciesspecificity.

Inhibition of β-Secretase in Vivo via Antibody Binding to Unique Loops (D and F) of BACE1

“…40, and materials were provided by Eli Lilly. Briefly, hBACE1:Fc was diluted into 1 nM in reaction buffer (50 mM ammonium acetate pH 4.6, 1 mg/ml BSA and 0.6% Triton X-100).…”

Robust Central Reduction of Amyloid-β in Humans with an Orally Available, Non-Peptidic β-Secretase Inhibitor

“…The MBP-C125Swe assay was conducted essentially as described by Sinha et al (1999). The 7-methoxycoumarin-4-yl-acetyl (mca) FRET assay for BACE2 was conducted essentially as described by Yang et al (2004). Human liver cathepsin D was purchased from Calbiochem (catalog #219041) and used as a source of enzyme for cathepsin D assays following the instructions of the manufacturer.…”

“…Human BACE2 was cloned, expressed, and purified essentially as described by Yang et al (2004). The MBP-C125Swe assay was conducted essentially as described by Sinha et al (1999).…”

“…Human BACE1 was cloned, expressed, and purified essentially as described by Yang et al (2004) and assayed using a synthetic peptide in a FRET format or using a MBP-C125Swe polypeptide substrate assay essentially as described by Yang et al (2004) or Sinha et al (1999), respectively.…”

Establishing the Relationship between In Vitro Potency, Pharmacokinetic, and Pharmacodynamic Parameters in a Series of Orally Available, Hydroxyethylamine-Derived β-Secretase Inhibitors

“…The extracellular domain of human BACE1 was expressed in CHO cells as described previously (Vassar et al, 1999). The enzymatic activity of recombinant BACE1 was measured by using a fluorescence resonance energy transfer (FRET) assay according to published methods (Yang et al, 2004). The substrate was a synthetic peptide that contained the BACE1 cleavage site (Turner et al, 2001) and fluorophore and quencher dyes at the termini of the peptide.…”