Identification and Expression of the cDNA-encoding Human Mesotrypsin(ogen), an Isoform of Trypsin with Inhibitor Resistance

Abstract

The cDNA encoding a novel isoform of human trypsinogen was identified. The isoelectric points of the proenzyme and active forms calculated from the deduced amino acid sequence are consistent with those of mesotrypsin(ogen), known to be an inhibitor-resistant trypsin isoform. The cDNA attached with a bacterial signal peptide sequence was expressed in Escherichia coli. The recombinant proenzyme purified from periplasm showed enterokinase-dependent activation similar to a major isoform of human trypsinogen. The enzyme was far less inhibited by trypsin inhibitors such as soybean trypsin inhibitor, aprotinin, or pancreatic secretory trypsin inhibitor than the control trypsin. A gel filtration assay showed that the enzyme and aprotinin did not form a stable complex. It is noteworthy that the amino acid at position 198, which is in close vicinity to the active Ser, is Arg while those of other major trypsins are all Gly. It is concluded that the cloned cDNA encodes human mesotrypsinogen, a unique isoform of trypsinogen with inhibitor resistance.Pancreatic trypsin is a key enzyme, which leads to activation of a number of pancreatic digestive proenzymes including chymotrypsinogens, procarboxypeptidases, proelastases, and prophospholipase A 2 as well as trypsinogens themselves. Three different trypsinogens, which show unique isoelectric points (pI), have been described in human pancreatic juice using twodimensional isoelectric focusing/SDS-PAGE.

Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites

“…Trypsin IV is resistant to polypeptide trypsin inhibitors, such as SBTI (18). Moreover, trypsin IV can degrade and inactivate these trypsin inhibitors (19,34,(43)(44)(45). In contrast to trypsin I, trypsin IV resists autoactivation and autolysis (43).…”

“…SBTI forms a stable, enzymatically inactive complex with trypsin I and also inhibits other proteases, including chymotrypsin, plasmin, and plasma kallikreins. In contrast, trypsin IV is resistant to polypeptide trypsin inhibitors, such as SBTI, which it also degrades (18,19,22,34). Therefore, SBTI should inhibit proteolytic activation of ENaC by trypsin I but not by trypsin IV.…”

“…The PRSS1 and PRSS2 genes are located on chromosome 7q34 and encode trypsinogen I and trypsinogen II, respectively. The PRSS3 gene, which is localized on chromosome 9p11.2, encodes mesotrypsinogen and a splice variant referred to as trypsinogen IV (17)(18)(19). After cleavage/activation by enteropeptidase (enterokinase), both precursor proteins result in the same active protease, which is referred to as mesotrypsin or trypsin IV.…”

Early events in acute pancreatitis

“…Although experimentally not demonstrated yet, logic would suggest that other pancreatic proteases might participate in a similar protective mechanism. In humans, mesotrypsin has been discussed as a candidate for this function [65,66]. The human pancreas secretes three isoforms of trypsinogen, encoded by PRSS1, 2 and 3.…”

Sequence and Conformational Specificity in Substrate Recognition

“…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).…”