The alternatively spliced Kunitz protease inhibitor domain alters amyloid beta protein precursor processing and amyloid beta protein production in cultured cells

The insoluble amyloid deposited extracellularly in the brains of patients with Alzheimer's disease (AD) is composed of amyloid beta protein, a similar to 4-kDa secreted protein that is derived from a set of large proteins collectively referred to as the amyloid beta protein precursor (beta APP). During normal processing the beta APP is cleaved by beta secretase, producing a large NH2-terminal secreted derivative (sAPP beta) and a COOH-terminal fragment beginning at A beta 1, which is subsequently cleaved by gamma secretase releasing secreted A beta. Most secreted A beta is A beta 1-40, but similar to 10% of secreted A beta is A beta 1-42. Alternative beta APP cleavage by alpha secretase produces a slightly longer NH2-terminal secreted derivative (sAPP alpha) and a COOH-terminal fragment beginning at A beta 17, which is subsequently cleaved by gamma secretase releasing a similar to 3-kDa secreted form of A beta (P3), Several of the beta APP 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 A beta, we compared A beta production in transfected cells expressing human KPI-containing beta APP751 or KPI-free beta APP695. We focused on A beta s ending at A beta 42 because these forms appear to be most relevant to AD. Using specific sandwich enzyme-linked immunosorbent assays, we analysed full-length A beta 1-42 and total A beta ending at A beta 42 (A beta 1-42 + P3(42)). In addition, we analysed the large secreted derivatives produced by alpha secretase (sAPP alpha) and beta secretase (sAPP beta). In mouse teratocarcinoma (P19) cells expressing beta APP695 or beta APP751, expression of the KPI-containing beta APP751 resulted in the secretion of a lower percentage of P3(42) and sAPP alpha and a correspondingly higher percentage of A beta 1-42 and sAPP beta. 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 A beta are produced. Thus, in human brain and in animal models of AD, the amount of KPI-containing beta APP produced may be an important factor influencing A beta deposition.