Structural Basis for Accelerated Cleavage of Bovine Pancreatic Trypsin Inhibitor (BPTI) by Human Mesotrypsin

Abstract

Human mesotrypsin is an isoform of trypsin that displays unusual resistance to polypeptide trypsin inhibitors and has been observed to cleave several such inhibitors as substrates. Whereas substitution of arginine for the highly conserved glycine 193 in the trypsin active site has been implicated as a critical factor in the inhibitor resistance of mesotrypsin, how this substitution leads to accelerated inhibitor cleavage is not clear. Bovine pancreatic trypsin inhibitor (BPTI) forms an extremely stable and cleavage-resistant complex with trypsin, and thus provides a rigorous challenge of mesotrypsin catalytic activity toward polypeptide inhibitors. Here, we report kinetic constants for mesotrypsin and the highly homologous (but inhibitor sensitive) human cationic trypsin, describing inhibition by, and cleavage of BPTI, as well as crystal structures of the mesotrypsin-BPTI and human cationic trypsin-BPTI complexes. We find that mesotrypsin cleaves BPTI with a rate constant accelerated 350-fold over that of human cationic trypsin and 150,000-fold over that of bovine trypsin. From the crystal structures, we see that small conformational adjustments limited to several side chains enable mesotrypsin-BPTI complex formation, surmounting the predicted steric clash introduced by Arg-193. Our results show that the mesotrypsin-BPTI interface favors catalysis through (a) electrostatic repulsion between the closely spaced mesotrypsin Arg-193 and BPTI Arg-17, and (b) elimination of two hydrogen bonds between the enzyme and the amine leaving group portion of BPTI. Our model predicts that these deleterious interactions accelerate leaving group dissociation and deacylation.There are three human trypsins encoded by different genes; cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2) are located at proximal loci on chromosome 7q35, while mesotrypsinogen (PRSS3) is found on chromosome 9p13 (1). All three isoforms are secreted as digestive zymogens in the pancreas and activated by enteropeptidase in the duodenum (2). A differentially spliced form of mesotrypsinogen termed trypsinogen 4, transcribed from an alternative promoter (3) and utilizing an unconventional CUG translation initiation codon (4), is highly expressed in brain tissue (5) and in some epithelial cell lines (6) and tumors (7). The two zymogen forms differ only at the N terminus, and processing of either form by removal of the prodomain results in active mesotrypsin of identical amino acid sequence (3). Trypsinogen 4 lacks a recognizable signal sequence and it is not known whether or how the enzyme might be secreted, though there is some evidence for processing of the prodomain and deposition of activated mesotrypsin in the extracellular neuronal matrix (8).The most striking characteristic of mesotrypsin is its unique resistance to polypeptide trypsin inhibitors (9, 10). Canonical trypsin inhibitors feature characteristic binding loops that bind to the trypsin active site extremely tightly, mimicking a substrate, yet are cleaved extremely slowly (11-13). Mesot...

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

“…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). Purified R117H, S195A trypsinogen (30 mg) was proteolytically processed by 12 g of bovine enteropeptidase (Roche Applied Science) in 80 ml of 100 mM HEPES, pH 8.0, 1 mM CaCl 2 for 5 h at 37°C.…”

“…Working stocks of enzyme, substrate, and inhibitors were prepared for competitive inhibition studies as described previously (11). Assays were conducted at 37°C in a Varian Cary-100 spectrophotometer equipped with a multicell transporter and a circulating water bath.…”

“…Production of Recombinant Human Proteases and APPI-Recombinant human cationic trypsinogen and mesotrypsinogen were expressed in Escherichia coli, isolated from inclusion bodies, refolded, purified, and activated with bovine enteropeptidase as described previously (11). Cationic trypsin and mesotrypsin concentrations were quantified by active-site titration using 4-nitrophenyl 4-guanidinobenzoate (Sigma) (32).…”

“…Dialyzed APPI samples were chromatographed on Q-Sepharose FF (GE Healthcare) and eluted with a gradient of 0 -100% buffer B (50 mM Tris, pH 7.8, 1 M NaCl) and then further purified on a trypsin affinity column and eluted with a gradient of 0 -100% buffer B (150 mM HCl). APPI concentrations were determined by titration with bovine trypsin (Sigma) as described previously (11).…”

“…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).…”