Biochem. J. (2003) 372, 227–233 (Printed in Great Britain) 227
Purification and identification of a binding protein for pancreatic secretory trypsin inhibitor: a novel role of the inhibitor as an anti-granzyme A Satoshi TSUZUKI*1,2,Yoshimasa KOKADO*1, Shigeki SATOMI*, Yoshie YAMASAKI*, Hirofumi HIRAYASU*, Toshihiko IWANAGA† and Tohru FUSHIKI* *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, and †Laboratory of Anatomy, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18-Nishi 9, Kita-ku, Sapporo 060-0818, Japan
Pancreatic secretory trypsin inhibitor (PSTI) is a potent trypsin of GzmA-expressing intraepithelial lymphocytes in the rat small inhibitor that is mainly found in pancreatic juice. PSTI has been intestine. We concluded that the PSTI-binding protein isolated shown to bind specifically to a protein, distinct from trypsin, on from the dispersed cells is GzmA that is produced in the the surface of dispersed cells obtained from tissues such as small lymphocytes of the tissue. The rGzmA hydrolysed the N-α- intestine. In the present study, we affinity-purified the binding benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT), and the BLT protein from the 2 % (w/v) Triton X-100-soluble fraction of hydrolysis was inhibited by PSTI. Sulphated glycosaminoglycans, dispersed rat small-intestinal cells using recombinant rat PSTI. such as fucoidan or heparin, showed almost no effect on the Partial N-terminal sequencing of the purified protein gave a inhibition of rGzmA by PSTI, whereas they decreased the inhi- sequence that was identical with the sequence of mouse granzyme bition by antithrombin III. In the present paper, we propose a novel A (GzmA), a tryptase produced in cytotoxic lymphocytes. We role of PSTI as a GzmA inhibitor. confirmed the formation of an affinity-cross-linked complex between 125I-labelled PSTI and recombinant rat GzmA (rGzmA). Keywords: affinity purification, glycosaminoglycan, granzyme In situ hybridization and immunostaining revealed the existence A, lymphoid tissue, pancreatic secretory trypsin inhibitor.
INTRODUCTION is distinct from trypsin. However, the chemical structure, the production site and the role of the specific binding remain to Pancreatic secretory trypsin inhibitor (PSTI), initially found by be investigated. Kazal et al. [1], is mainly produced in the acinar cells of the The purpose of the present study was to identify the binding pancreas and is secreted into pancreatic juice. PSTI is a small protein for PSTI. We affinity-purified the binding protein with a protein with a molecular mass of about 6.5 kDa [2]. Its role has recombinant PSTI from small intestine, where the binding site been postulated to be the prevention of inadvertent proteolysis in is the most abundant [7]. Partial N-terminal sequencing of the the pancreas or pancreatic duct caused by premature activation purified protein revealed that it is identical with granzyme A of trypsinogen [2]. The inhibitory specificity is very narrow, (GzmA), a lymphocyte tryptase [8]. The present study documents with strong inhibitory capacity against pancreatic trypsin, a potential role of PSTI as the tryptase inhibitor. butwith very little or no inhibitory capacity against various proteases with trypsin-like specificity [3]. Considerable amounts of PSTI have also been detected in a variety of extrapancreatic EXPERIMENTAL tissues [4]. In addition, Ogawa and co-workers [5,6] have found elevated serum immunoreactive PSTI content in patients with Materials severe inflammation and tissue destruction, where almost no Male Wistar rats were purchased from Japan Shizuoka Laboratory trypsin activity in blood has been detected. These previous results Center (Hamamatsu, Japan). Trypsin (type III, from bovine suggest that PSTI has other important functions outside the pancreas), γ -globulin, soybean trypsin inhibitor (type IS), pancreas. N-α-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) [9], We previously found a specific binding protein for PSTI on the 5,5 -dithiobis-(2-nitrobenzoic acid) and fucoidan (from Fucus surface of dispersed cells obtained from small intestine, colon, vesiculosus)werepurchased from Sigma (St. Louis, MO, U.S.A.). liver, stomach and kidney [7]. Autoradiography of the affinity- Antithrombin III (ATIII), chondroitin sulphate A, and heparin cross-linked complex of the 125I-labelled PSTI and the binding were purchased from Wako Pure Chemicals Co. (Tokyo, Japan). protein indicated a molecular mass of about 33 kDa and 53 kDa An anti-GzmA polyclonal antibody (D-15) was purchased from after SDS/PAGE (12 % gels), under reducing and non-reducing Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.). conditions respectively, suggesting that the binding molecule Ni2+-charged resin was purchased from CytoSignal Research
Abbreviations used: ATIII, antithrombin III; BLT, N-α-benzyloxycarbonyl-L-lysine thiobenzyl ester; BS3,bis(sulphosuccinimidyl) suberate; CCK, cholecystokinin; CTL, cytotoxic T-lymphocyte; GdmCl, guanidinium chloride; GzmA, granzyme A; GzmB, granzyme B; IL, interleukin; NK cells, naturalkiller cells; PSTI, pancreatic secretory trypsin inhibitor; rGzmA, rat GzmA; rhPSTI, recombinant human PSTI; rrPSTI–His, recombinant rat PSTI with C-terminal polyhistidine tag. 1 Both authors contributed equally to this work. 2 To whom correspondence should be addressed (e-mail [email protected]). The nucleotide sequence data of the rat granzyme A are available in the DDBJ, EMBL, GenBank R and GSDB Nucleotide Sequence Databases under the accession number AB082125.