Purification and cloning of amyloid precursor protein beta-secretase from human brain.
Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (Abeta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the Abeta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of Abeta. The pathogenic mutation K670M671-->N670L671 at the beta-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.
Sinha
S
S
Elan Pharmaceuticals, South San Francisco, California 94080, USA. ssinha@elanpharama.com
Anderson
J P
JP
Barbour
R
R
Basi
G S
GS
Caccavello
R
R
Davis
D
D
Doan
M
M
Dovey
H F
HF
Frigon
N
N
Hong
J
J
Jacobson-Croak
K
K
Jewett
N
N
Keim
P
P
Knops
J
J
Lieberburg
I
I
Power
M
M
Tan
H
H
Tatsuno
G
G
Tung
J
J
Schenk
D
D
Seubert
P
P
Suomensaari
S M
SM
Wang
S
S
Walker
D
D
Zhao
J
J
McConlogue
L
L
John
V
V
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D006224:Cricetinae; D010450:Endopeptidases; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014162:Transfection
D016564:Amyloid beta-Protein Precursor; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509284:BACE2 protein, human; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse