A Possible Role for Cathepsins D, E, and B in the Processing of β-amyloid Precursor Protein in Alzheimer's Disease

Abstract

Formation of the 4-kDa peptides, which are essential constituents of the extracellular plaques in Alzheimer's disease, involves the sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. The carboxy-terminal 99-amino-acid peptide which is liberated from APP by β-secretase was used as a potential native substrate of the γ-secretase(s). With the addition of an initiator Met and a FLAG sequence at the C-terminus (β5APP100-FLAG), it was expressed in Escherichia coli under the control of the T7 promoter. The preferred site(s) of cleavage in the N-terminal 40-amino-acid β-amyloid peptide and βAPP100-FLAG by potential γ-secretase(s) were rapidly identified using matrix-assisted laser-desorption/ionization time-of-flight mass spectroscopy in addition to peptide mapping followed by protein sequence analysis. Since γ-secretases seem to be active at acidic pH, three cathepsins (D, E and B) were selected for testing. Studies using different detergents indicated that the cleavage preference of cathepsin D for the βAPP100-FLAG is highly dependent on the surfactant used to solubilize this substrate. All three cathepsins were found to be capable of catabolizing both β-amyloid peptides and the βAPP100-FLAG. As cathepsin D was found to cleave the βAPP100-FLAG in the vicinity of the C-terminus of the β-amyloid peptides and cathepsin B has a high carboxypeptidase activity at low pH, the possibility cannot be excluded that cathepsins D and B are involved in the amyloidogenic processing of APP.

Abbreviations.

APP

amyloid precursor protein

βA-(1 -40)-peptide

β-amyloid peptide containing 40 amino acids

βAPP99

C-terminal 99 amino acids of APP

βAPP100

C-terminal 100 amino acids of APP

βAPP100-FLAG

C-terminal 100 amino acids of APP plus an additional FLAG sequence consisting of amino acids DYKDDDDK at the C-terminus

Brij 30

4-lauryl ether

Brij 35

23 lauryl ether

HedMe₃NBr

hexadecyl tnmethylammonium bromide

LC-MS

on-line liquid chromatography and electrospray mass spectroscopy

MALDI-TOF

matrix-assisted laser-desorption/ionization time-of-flight mass spectroscopy

mlz

mass/charge ratio

RP

reverse phase

Enzymes.

 

Cathepsin D (EC 3.4.23.5)

 

cathepsin E (EC 3.4.23.34)

 

cathepsin B (EC 3.4.22.1)