Abstract
The amyloid precursor protein (APP) is a ubiquitously expressed transmembrane adhesion protein and the progenitor of amyloid-beta peptides. The major splice isoforms of APP expressed by most tissues contain a Kunitz protease inhibitor domain; secreted APP containing this domain is also known as protease nexin 2 and potently inhibits serine proteases, including trypsin and coagulation factors. The atypical human trypsin isoform mesotrypsin is resistant to inhibition by most protein protease inhibitors and cleaves some inhibitors at a substantially accelerated rate. Here, in a proteomic screen to identify potential physiological substrates of mesotrypsin, we find that APP/protease nexin 2 is selectively cleaved by mesotrypsin within the Kunitz protease inhibitor domain. In studies employing the recombinant Kunitz domain of APP (APPI), we show that mesotrypsin cleaves selectively at the Arg(15)-Ala(16) reactive site bond, with kinetic constants approaching those of other proteases toward highly specific protein substrates. Finally, we show that cleavage of APPI compromises its inhibition of other serine proteases, including cationic trypsin and factor XIa, by 2 orders of magnitude. Because APP/protease nexin 2 and mesotrypsin are coexpressed in a number of tissues, we suggest that processing by mesotrypsin may ablate the protease inhibitory function of APP/protease nexin 2 in vivo and may also modulate other activities of APP/protease nexin 2 that involve the Kunitz domain.
Highlights
Mesotrypsin is a human trypsin encoded by the PRSS3 gene found on chromosome 9p13 [1]
A prominent exception, for which mesotrypsin appears to possess enhanced catalytic capability compared with its proteolytic siblings, is in the cleavage of canonical protease inhibitors [7, 11]
We have identified the mesotrypsin cleavage site as the Arg15-Ala16 reactive site bond of the amyloid precursor protein (APP) Kunitz protease inhibitor domain, and we have shown that this cleavage drastically diminishes the effectiveness of the APP Kunitz domain as an inhibitor of serine proteases
Summary
Cell Culture—WPE1 NB11 and LNCaP cells were obtained from ATCC. Both are malignant, tumorigenic cell lines of human prostate epithelial origin. Trypsin Affinity Chromatography of Conditioned Cell Culture Medium—The recombinant, catalytically inactive R117H,S195A mutant of human cationic trypsinogen was expressed, refolded, and purified as described previously [11]. Pooled extracts were concentrated and brought up in 0.1% formic acid for protein identification by nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) analysis using a ThermoFinnigan LTQ Orbitrap hybrid mass spectrometer coupled to an Eksigent nanoLC-two-dimensional HPLC system. APPI concentrations were determined by titration with bovine trypsin (Sigma) as described previously [11]. Mesotrypsin or cationic trypsin, APPI, and hydrolysis products were resolved on a 50 ϫ 2.0-mm Jupiter 4 90 Å C12 column (Phenomenex) with a gradient of 0 –100% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 0.6 ml/min over 50 min. Initial rates were obtained by linear regression using a minimum of six data points within the initial linear phase of the reaction; the hydrolysis rates reported represent the average of three independent experiments
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