Tsuzuki Satoshi, Kokado Yoshimasa, Satomi Shigeki, Yamasaki Yoshie, Hirayasu Hirofumi, Iwanaga Toshihiko, Fushiki Tohru
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.
Biochem J. 2003 May 15;372(Pt 1):227-33. doi: 10.1042/BJ20021891.
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.
胰腺分泌型胰蛋白酶抑制剂（PSTI）是一种强效胰蛋白酶抑制剂，主要存在于胰液中。已表明PSTI能特异性结合从小肠等组织获得的分散细胞表面一种不同于胰蛋白酶的蛋白质。在本研究中，我们使用重组大鼠PSTI从分散的大鼠小肠细胞2%（w/v） Triton X-100可溶性组分中亲和纯化该结合蛋白。对纯化蛋白的部分N端测序得到的序列与小鼠颗粒酶A（GzmA）相同，GzmA是一种由细胞毒性淋巴细胞产生的类胰蛋白酶。我们证实了（125）I标记的PSTI与重组大鼠GzmA（rGzmA）之间形成了亲和交联复合物。原位杂交和免疫染色显示大鼠小肠中存在表达GzmA的上皮内淋巴细胞。我们得出结论，从分散细胞中分离出的PSTI结合蛋白是组织淋巴细胞中产生的GzmA。rGzmA能水解N-α-苄氧羰基-L-赖氨酸硫代苄酯（BLT），且BLT水解受到PSTI的抑制。硫酸化糖胺聚糖，如岩藻依聚糖或肝素，对PSTI抑制rGzmA几乎没有影响，而它们能降低抗凝血酶III的抑制作用。在本文中，我们提出了PSTI作为GzmA抑制剂的新作用。
