A possible role for cathepsins D, E, and B in the processing of beta-amyloid precursor protein in Alzheimer's disease.
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 beta- and gamma-secretases. The carboxy-terminal 99-amino-acid peptide which is liberated from APP by beta-secretase was used as a potential native substrate of the gamma-secretase(s). With the addition of an initiator Met and a FLAG sequence at the C-terminus (betaAPP100-FLAG), it was expressed in Escherichia coli under the control of the T7 promotor. The preferred site(s) of cleavage in the N-terminal 40-amino-acid beta-amyloid peptide and betaAPP100-FLAG by potential gamma-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 gamma-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 betaAPP100-FLAG is highly dependent on the surfactant used to solubilize this substrate. All three cathepsins were found to be capable of catabolizing both beta-amyloid peptides and the betaAPP100-FLAG. As cathepsin D was found to cleave the betaAPP100-FLAG in the vicinity of the C-terminus of the beta-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.
Mackay
E A
EA
Marion Merrell Research Institute, Strasbourg, France.
Ehrhard
A
A
Moniatte
M
M
Guenet
C
C
Tardif
C
C
Tarnus
C
C
Sorokine
O
O
Heintzelmann
B
B
Nay
C
C
Remy
J M
JM
Higaki
J
J
Van Dorsselaer
A
A
Wagner
J
J
Danzin
C
C
Mamont
P
P
D000544:Alzheimer Disease / Q000378:metabolism*; D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002401:Cathepsin B / Q000378:metabolism*; D002402:Cathepsin D / Q000378:metabolism*; D020059:Cathepsin E; D002403:Cathepsins / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009842:Oligopeptides; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D016564:Amyloid beta-Protein Precursor; D017931:DNA Primers; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; C085706:FLAG peptide; D002403:Cathepsins; D002401:Cathepsin B; D020059:Cathepsin E; D002402:Cathepsin D