Human mesotrypsin exhibits restricted S1′ subsite specificity with a strong preference for small polar side chains

Although principally produced by the pancreas to degrade dietary proteins in the intestine, trypsins are also expressed in the nervous system and in epithelial tissues, where they have diverse actions that could be mediated by protease-activated receptors (PARs). We examined the biological actions of human trypsin IV (or mesotrypsin) and rat p23, inhibitor-resistant forms of trypsin. The zymogens trypsinogen IV and pro-p23 were expressed in Escherichia coli and purified to apparent homogeneity. Enteropeptidase cleaved both zymogens, liberating active trypsin IV and p23, which were resistant to soybean trypsin inhibitor and aprotinin. (trypsin IV and p23) mice. Trypsin IV and p23 caused thermal hyperalgesia and mechanical allodynia and hyperalgesia in mice, and these effects were absent in PAR 2 ؊/؊ mice but maintained in PAR 1 ؊/؊ mice. Thus, trypsin IV and p23 are inhibitor-resistant trypsins that can cleave and activate PARs, causing PAR 1 -and PAR 2 -dependent inflammation and PAR 2 -dependent hyperalgesia.

Section: Discussionsupporting

confidence: 51%

Trypsin IV or Mesotrypsin and p23 Cleave Protease-activated Receptors 1 and 2 to Induce Inflammation and Hyperalgesia

Knecht

Cottrell

Amadesi

et al. 2007

“…Human SPINK1 was expressed in HEK 293T cells and purified from the conditioned medium using a bovine trypsin affinity column. Ecotin and human ␣1-antitrypsin were expressed in Escherichia coli and purified as described previously (15)(16)(17). Soybean trypsin inhibitor was from Fluka and was further purified on a bovine trypsin affinity column.…”

Section: Methodsmentioning

confidence: 99%

Intracellular Autoactivation of Human Cationic Trypsinogen Mutants Causes Reduced Trypsinogen Secretion and Acinar Cell Death

Kereszturi

Sahin–Tóth

2009

Mutations in the activation peptide of human cationic trypsinogen have been found in patients with chronic pancreatitis. Previous biochemical studies demonstrated that mutations p.D19A, p.D22G, and p.K23R strongly stimulate trypsinogen autoactivation. In the present study, we characterized the cell biological effects of these mutants using human embryonic kidney 293T and AR42J rat acinar cells. We found that relative to wild-type trypsinogen, secretion of the mutants from transfected cells was markedly decreased. This apparent secretion defect was completely rescued by inhibition of autoactivation via (1)

“…Although mesotrypsin shows high sequence homology with other trypsins, its functional properties are very different. Mesotrypsin is highly resistant to inhibition by many polypeptide trypsin inhibitors (39 -41), although it is readily inhibited by serpin-type inhibitors with Arg in the reactive loop (42,43). Although it cleaves peptide anilide model substrates with kinetic constants similar to other trypsins (44 -46), it displays reduced or no activity toward several specific protein substrates of other trypsins (44,(47)(48)(49) and, unlike other trypsins, is incapable of autoactivation (44).…”

mentioning

confidence: 99%

Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

Salameh

Soares

Navaneetham

et al. 2010

An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P 1 (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P 2 favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P 1 and P 2 substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin⅐APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.