The Amyloid Precursor Protein/Protease Nexin 2 Kunitz Inhibitor Domain Is a Highly Specific Substrate of Mesotrypsin

Abstract

The amyloid precursor protein (APP) is a ubiquitously expressed transmembrane adhesion protein and the progenitor of amyloid-␤ 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.Mesotrypsin is a human trypsin encoded by the PRSS3 gene found on chromosome 9p13 (1). Normal expression of PRSS3 is restricted to pancreas, brain, and, to a lesser extent, small intestine and colon (2-4); additionally, PRSS3 appears to be transcriptionally up-regulated with cancer progression in epithelial cancers, including lung (5), colon (6), and prostate. Mesotrypsin exhibits substantially different specificity from other trypsins toward protein substrates. It fails to activate pancreatic zymogens and also shows reduced capacity to degrade trypsinogens (7). Compared with other trypsins, it is significantly compromised in its ability to cleave protease-activated receptors (8 -10). Despite limited activity toward these classic trypsin substrates, mesotrypsin displays enhanced catalytic activity compared with other trypsins in the cleavage of certain specific protein substrates, most notably several canonical protease inhibitors (7, 11).The "canonical" inhibitors of serine proteases, named for a protease-binding loop of highly characteristic backbone conformation (12, 13), fulfill the paradoxical function of binding to a protease in a substrate-like manner yet acting as an inhibitor rather than an ordinary substrate. These inhibitors, representing at least 18 different convergently evolved protein families (14, 15), inhibit their cognate proteases via the "Laskowski mechanism," in which inhibitors act as highly specific, limited proteolysis substrates for target enzymes (14, 16). They bind so as to position a specific peptide bond, the "reactive si...

Sequence and Conformational Specificity in Substrate Recognition

“…Surprisingly, we found considerable differences between BPTI and APPI in their relative susceptibility to cleavage by mesotrypsin. Whereas APPI was efficiently cleaved by mesotrypsin in seconds, with kinetics resembling those of a good substrate (15), BPTI behaved as a weak temporary inhibitor of mesotrypsin and was only gradually cleaved over the course of hours (22). Mutagenesis studies targeting the canonical binding loops of APPI and BPTI revealed that these differences were not solely determined by the cleavage site sequences, but rather a large proportion of the superior proteolytic stability of BPTI was endemic to the protein scaffold (23), suggesting that the resistance of different Kunitz domains to proteolysis is quite variable.…”

“…PRSS3 transcripts, primarily encoding trypsinogen 4, are also expressed in many epithelial cells and cancers, and functional studies likewise provide evidence for extracellular roles for active mesotrypsin in tumor progression of breast (8), pancreatic (9), and prostate cancers (10,11). An unusual feature of mesotrypsin is its extreme resistance to endogenous proteinaceous trypsin inhibitors (1,12,13) and its ability to digest some of these inhibitors as substrates (6,14,15). We and others have hypothesized that physiological functions of mesotrypsin may derive from its ability to degrade protein protease inhibitors, for example by digesting plant-derived serine protease inhibitors, such as soy bean trypsin inhibitor in the diet (14), or by degrading the lymphoepithelial Kazal-type inhibitor LEKTI to promote desquamation (6).…”

“…Both of these potent trypsin inhibitors are bound orders of magnitude more weakly and cleaved orders of magnitude more rapidly by mesotrypsin than by typical trypsins (15,22). Furthermore, the canonical conformation of the binding loop was found to be critical for recognition as a substrate by mesotrypsin, because an unstructured peptide containing the same reactive site sequence as APPI was not bound or cleaved by mesotrypsin (15). Surprisingly, we found considerable differences between BPTI and APPI in their relative susceptibility to cleavage by mesotrypsin.…”

“…For final kinetics studies using bikunin-KD2, HAI2-KD1, TFPI1-KD1, TFPI1-KD2, and TFPI2-KD1 as substrates, aliquots for HPLC analysis were withdrawn at periodic intervals, adjusted to 6 M urea and 2 mM DTT, incubated for 10 min at 37°C, quenched by acidification with HCl to pH 1, and then frozen at Ϫ20°C until analyzed, as reported previously for hydrolysis studies with BPTI (22). For APLP2-KD, reactions were carried out similarly, but aliquots for analysis were not subjected to reduction/denaturation with DTT/urea, as described previously for hydrolysis studies with APPI (15). Enzyme, intact Kunitz domains, and hydrolysis products were resolved on a 50 ϫ 2.0 -33 Jupiter 4 90-Å C 12 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.…”

“…Recombinant Kunitz domains were expressed and purified from the methylotropic yeast P. pastoris under control of the alcohol oxidase (AOX1) promotor, following protocols described previously for BPTI (23,25) and APPI (15,26) with minor modifications. Briefly, expression cultures grown in BMMY medium (buffered medium containing methanol and yeast nitrogen base) at 30°C were harvested after 72 h. The supernatant was subjected to ammonium sulfate precipitation (95% saturation) at room temperature.…”