Purification and identification of a binding protein for pancreatic secretory trypsin inhibitor: a novel role of the inhibitor as an anti-granzyme A.
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.
Tsuzuki
Satoshi
S
Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan. tkmone@kais.kyoto-u.ac.jp
Kokado
Yoshimasa
Y
Satomi
Shigeki
S
Yamasaki
Yoshie
Y
Hirayasu
Hirofumi
H
Iwanaga
Toshihiko
T
Fushiki
Tohru
T
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism*; D053804:Granzymes; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007421:Intestine, Small / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017208:Rats, Wistar; D012697:Serine Endopeptidases / Q000378:metabolism*; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000378:metabolism*
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D014359:Trypsin Inhibitor, Kazal Pancreatic; D053804:Granzymes; D012697:Serine Endopeptidases