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

The insoluble amyloid deposited extracellularly in the brains of patients with Alzheimer's disease (AD) is composed of amyloid β protein, a ~4-kDa secreted protein that is derived from a set of large proteins collectively referred to as the amyloid β protein precursor (βAPP). During normal processing the βAPP is cleaved by β secretase, producing a large NH₂-terminal secreted derivative (sAPPβ) and a COOH-terminal fragment beginning at Aβ1, which is subsequently cleaved by γ secretase releasing secreted Aβ. Most secreted Aβ is Aβ1-40, but ~10% of secreted Aβ is Aβ1-42. Alternative βAPP cleavage by α secretase produces a slightly longer NH₂-terminal secreted derivative (sAPPα) and a COOH-terminal fragment beginning at Aβ17, which is subsequently cleaved by γ secretase releasing a ~3-kDa secreted form of Aβ (P3). Several of the βAPP 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 Aβ, we compared Aβ production in transfected cells expressing human KPI-containing βAPP751 or KPI-free βAPP695. We focused on Aβs ending at Aβ42 because these forms appear to be most relevant to AD. Using specific sandwich enzyme-linked immunosorbent assays, we analyzed full-length Aβ1-42 and total Aβ ending at Aβ42 (Aβ1- 42 + P3(42)). In addition, we analyzed the large secreted derivatives produced by α secretase (sAPPγ) and β secretase (sAPPβ). In mouse teratocarcinoma (P19) cells expressing βAPP695 or βAPP751, expression of the KPI-containing βAPP751 resulted in the secretion of a lower percentage of P3(42) and sAPPα and a correspondingly higher percentage of Aβ1-42 and sAPPβ. Similar results were obtained in human embryonic kidney (293) cells. These results indicate that expression of the KPI domain reduces a secretase cleavage so that less P3 and relatively more full-length Aβ are produced. Thus, in human brain and in animal models of AD, the amount of KPI-containing βAPP produced may be an important factor influencing Aβ deposition.