Enzyme specificity of proteinase inhibitor region in amyloid precursor protein of Alzheimer's disease: different properties compared with protease nexin I.
Senile plaques, often surrounded by abnormally grown neurites, are characteristic of Alzheimer's diseased brain. The core of the plaque is mainly composed of amyloid beta protein (beta-AP), two of whose three precursors (APP) have serine proteinase inhibitor regions (APPI). APPI derivatives containing 60, 72 or 88 amino-acid fragments (APPI-60, APPI-72 and APPI-88, respectively) of the longest APP were produced in COS-1 cell culture medium, with the APPI cDNA ligated to the signal sequence of tissue plasminogen activator. The secreted APPIs were purified by sequential acetone precipitation followed by affinity chromatography using immobilized trypsin. These three APPIs and O-glycosylation-site-mutated APPI showed similar inhibitory activity against trypsin, chymotrypsin and plasmin. The purified APPI-72 was found to inhibit trypsin (Ki = 1.1 x 10(-10) M) and chymotrypsin (Ki = 5.8 x 10(-9) M) most strongly, and to inhibit leukocyte elastase (Ki = 7.9 x 10(-7) M) and several blood coagulation proteinases (Ki = 0.46-12 x 10(-7) M), but not urokinase or thrombin. The observed inhibition pattern was quite different from that of protease nexin I, one of serine proteinase inhibitors possessing neurite outgrowth activity. This suggests that the physiological roles of APPI are different from those of protease nexin I, and that APPI could not cause aberrant growth of neurite into the plaque. The presence of APPI having strong inhibitory activity in the brain might lead to the formation of amyloid deposits by preventing complete degradation of APPs.
Kitaguchi
N
N
Life Science Research Laboratories, Asahi Chemical Industry Co., Shizuoka, Japan.
Takahashi
Y
Y
Oishi
K
K
Shiojiri
S
S
Tokushima
Y
Y
Utsunomiya
T
T
Ito
H
H
D000595:Amino Acid Sequence; D000682:Amyloid / Q000302:isolation & purification / Q000378:metabolism*; D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D004252:DNA Mutational Analysis; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D013379:Substrate Specificity
C545487:APP protein, human; D000682:Amyloid; D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D011480:Protease Inhibitors; D058255:Protease Nexins; D011956:Receptors, Cell Surface