Purification and identification of a binding protein for pancreatic secretory trypsin inhibitor: a novel role of the inhibitor as an anti-granzyme A

Abstract

Pancreatic secretory trypsin inhibitor (PSTI) is a potent trypsin inhibitor that is mainly found in pancreatic juice. PSTI has been shown to bind specifically to a protein, distinct from trypsin, on the surface of dispersed cells obtained from tissues such as small intestine. In the present study, we affinity-purified the binding protein from the 2% (w/v) Triton X-100-soluble fraction of dispersed rat small-intestinal cells using recombinant rat PSTI. Partial N-terminal sequencing of the purified protein gave a sequence that was identical with the sequence of mouse granzyme A (GzmA), a tryptase produced in cytotoxic lymphocytes. We confirmed the formation of an affinity-cross-linked complex between (125)I-labelled PSTI and recombinant rat GzmA (rGzmA). In situ hybridization and immunostaining revealed the existence of GzmA-expressing intraepithelial lymphocytes in the rat small intestine. We concluded that the PSTI-binding protein isolated from the dispersed cells is GzmA that is produced in the lymphocytes of the tissue. The rGzmA hydrolysed the N -alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT), and the BLT hydrolysis was inhibited by PSTI. Sulphated glycosaminoglycans, such as fucoidan or heparin, showed almost no effect on the inhibition of rGzmA by PSTI, whereas they decreased the inhibition by antithrombin III. In the present paper, we propose a novel role of PSTI as a GzmA inhibitor.

A major role for proteolytic activity and proteinase-activated receptor-2 in the pathogenesis of infectious colitis

“…Thus, rather than secreting a bacterial proteinase, C. rodentium infection in vivo induces host tissues to release PAR 2 -activating serine proteinases. Granzyme A and kallikrein B are both known to be inhibited by STI (36,37), and it is possible that the inhibition by STI of these proteinases could account for its antiinflammatory effects in vivo. It also needs to be recognized that human mesotrypsin (38) and trypsin IV (39) are not inhibited by STI, and, in fact, mesotrypsin degrades STI.…”

Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death

“…Extrapancreatic or blood PSTI might regulate extracellular activity of GzmA. Unlike the other two GzmA inhibitors, PSTI inhibits GzmA complexed to proteoglycans (115). It is still not clear whether any of these GzmA inhibitors are expressed in cytotoxic lymphocytes.…”

“…Extracellular GzmA complexed to proteoglycans is resistant to these two protease inhibitors (114). A recent study identified another GzmA inhibitor, pancreatic secretory trypsin inhibitor (PSTI), from pancreatic secretions (115). PSTI is found in the blood, particularly in patients with severe inflammation and tissue destruction (116,117).…”

Granzyme A and thrombin differentially promote the release of interleukin-8 from alveolar epithelial A549 cells

“…-charged resin (HisLink TM resin, Promega, Madison, WI, USA) were performed as described previously (Hirayasu et al 2005(Hirayasu et al , 2007(Hirayasu et al , 2008Tsuzuki et al 2003). In order to obtain the active form, the purified rGrA was incubated with 2.0 units/mL recombinant enterokinase (Novagen, Madison, WI, USA) for 18 h at 22°C.…”