Abstract
The insoluble amyloid deposited extracellularly in the brains of patients with Alzheimer's disease (AD) is composed of amyloid beta protein, a approximately 4-kDa secreted protein that is derived from a set of large proteins collectively referred to as the amyloid beta protein precursor (betaAPP). During normal processing the betaAPP is cleaved by beta secretase, producing a large NH2-terminal secreted derivative (sAPPbeta) and a COOH-terminal fragment beginning at Abeta1, which is subsequently cleaved by gamma secretase releasing secreted Abeta. Most secreted Abeta is Abeta1-40, but approximately 10% of secreted Abeta is Abeta1-42. Alternative betaAPP cleavage by alpha secretase produces a slightly longer NH2-terminal secreted derivative (sAPPalpha) and a COOH-terminal fragment beginning at Abeta17, which is subsequently cleaved by gamma secretase releasing a approximately 3-kDa secreted form of Abeta (P3). Several of the betaAPP isoforms that are produced by alternative splicing contain a 56-amino acid Kunitz protease inhibitor (KPI) domain known to inhibit proteases such as trypsin and chymotrypsin. To determine whether the KPI domain influences the proteolytic cleavages that generate Abeta, we compared Abeta production in transfected cells expressing human KPI-containing betaAPP751 or KPI-free betaAPP695. We focused on Abetas ending at Abeta42 because these forms appear to be most relevant to AD. Using specific sandwich enzyme-linked immunosorbent assays, we analyzed full-length Abeta1-42 and total Abeta ending at Abeta42 (Abeta1-42 + P3(42)). In addition, we analyzed the large secreted derivatives produced by alpha secretase (sAPPalpha) and beta secretase (sAPPbeta). In mouse teratocarcinoma (P19) cells expressing betaAPP695 or betaAPP751, expression of the KPI-containing betaAPP751 resulted in the secretion of a lower percentage of P3(42) and sAPPalpha and a correspondingly higher percentage of Abeta1-42 and sAPPbeta. Similar results were obtained in human embryonic kidney (293) cells. These results indicate that expression of the KPI domain reduces alpha secretase cleavage so that less P3 and relatively more full-length Abeta are produced. Thus, in human brain and in animal models of AD, the amount of KPI-containing betaAPP produced may be an important factor influencing Abeta deposition.
Highlights
Characteristic pathological feature is the deposition of amyloid in senile plaques
Alzheimer’s disease (AD) amyloid is composed of amyloid  protein (A), a ϳ4-kDa secreted protein released from an ϳ120-kDa amyloid  protein precursor (APP) through cleavage by proteases referred to as secretases
The secreted form of the KPI-containing APP751 is identical to protease nexin II, a plasma serine protease inhibitor (21, 22), and it has been shown to inhibit proteases such as trypsin (23–25), chymotrypsin (22, 24, 25), protein; APP, amyloid  protein precursor; sAPP, secreted derivative of APP cleaved by  secretase; sAPP␣, secreted derivative of APP cleaved by ␣ secretase; KPI, Kunitz protease inhibitor; ELISA, enzymelinked immunosorbent assay; tresyl, 2,2,2-trifluoroethanesulfonyl; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
Summary
Cell Culture—Mouse teratocarcinoma P19 cells and human embryonic kidney 293 cells expressing APP695 and APP751 under the control of a cytomegalovirus enhancer and chick -actin promoter were prepared by transfection with modified pCXN2 vectors (28) constructed as described by Fukuchi et al.[2]. Radiolabeling Experiments—Subconfluent cells on 10-cm plates were labeled for 5 or 8 h with Tran35S-label (250 Ci/ml) in 5 ml of minimum essential medium deficient for methionine/cysteine. Radiolabeled A released into the medium was immunoprecipitated with Aspecific antibodies: R1280 For measurement of the APP synthesis rate, cells seeded at 2– 4 ϫ 105/well in six-well plates on the day prior to experimentation were labeled for 20 min with Tran35S-label (250 Ci/ml) in 0.5 ml of minimum essential medium deficient for methionine/cysteine. Using antibody to the APP COOH terminus, the radiolabeled APP was immunoprecipitated from lysates, separated on 10% Tris-Tricine gels, and quantitated by PhosphorImaging as described above. To assess sAPP␣ and sAPP, cells seeded at 2– 4 ϫ 105/well in six-well plates on the day prior to experimentation were labeled for 2.5–10 h as described above. The immunoprecipitated sAPPs were processed and quantitated as described above
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