Interaction of Protein C Inhibitor with the Type II Transmembrane Serine Protease Enteropeptidase

Abstract

The serine protease inhibitor protein C inhibitor (PCI) is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP) is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48×10 4 M −1 s −1 . Low molecular weight (LMWH) and unfractionated heparin (UFH) slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition) value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml). By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.

Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden

“…The eight amino acid sequence, 164 RKKRSTSA, containing the furin cleavage site (furin cleaves the peptide bond between R-S) in the mature wild-type PrAg protein (PrAg-WT) was replaced with the sequence 164 FTFRSARL (to create PrAg-PCIS) using an overlap PCR strategy. This substrate sequence was derived from a region of protein C inhibitor (PCI, SERPINA5 ), within the reactive center loop and close to the C-terminus, and is known to be cleaved by testisin [ 65 ], as we confirmed (Figure 1A ), as well as by other serine proteases [ 66 – 68 ]. The mutant and wild-type PrAg cDNAs were expressed in the non-virulent B. anthracis strain BH460, and the secreted PrAg proteins purified in high yield using established protocols [ 69 ].…”

Antithrombin controls tumor migration, invasion and angiogenesis by inhibition of enteropeptidase

“…Recently, it has been shown that protein C inhibitor (PCI) and, to a lesser extent, antithrombin, may regulate the function of enteropeptidase. However, PCI does not inhibit enteropeptidase in vivo 12 . In this study, we characterized the antithrombin-mediated inhibition of enteropeptidase and investigated the effects of antithrombin on tumor cell migration, invasion and angiogenesis.…”

A+-Helix of Protein C Inhibitor (PCI) Is a Cell-penetrating Peptide That Mediates Cell Membrane Permeation of PCI

“…It belongs to clade A of the serpin ( ser ine p rotease in hibitor) superfamily ( 1 ) and was initially identified in human plasma as an inhibitor of the anticoagulant serine protease activated protein C (aPC) ( 2 ). Subsequently, PCI was recognized to be an inhibitor of many other serine proteases including blood coagulation factors ( 3 ), fibrinolytic enzymes ( 4 , 5 ), tissue kallikrein ( 6 ), acrosin ( 7 ), hepatocyte growth factor activator ( 8 ), and enteropeptidase ( 9 ). Like other serpins, PCI inhibits its target proteases by forming covalent enzyme-serpin complexes ( 10 ).…”

“…In addition, glycosaminoglycans and phospholipids present on cell membranes ( 13 , 18 ) may not only mediate the permeation of PCI through membranes but may also bring PCI into close vicinity of cell membrane-associated serine proteases. To the best of our knowledge, there are only a few publications about the interaction of PCI with membrane-associated serine proteases ( 9 , 19 , 20 ). Among those reports, enteropeptidase is the only membrane-anchored serine protease whose interaction with PCI has been biochemically characterized ( 9 ).…”

“…To the best of our knowledge, there are only a few publications about the interaction of PCI with membrane-associated serine proteases ( 9 , 19 , 20 ). Among those reports, enteropeptidase is the only membrane-anchored serine protease whose interaction with PCI has been biochemically characterized ( 9 ).…”