Protease inhibitors are widely distributed in the skin mucus of fish. The pufferfish, family Tetraodontidae, has high antitryptic activity among fish. In this study, we isolated and characterized a protease inhibitor from the skin mucus of the pufferfish Takifugu rubripes. The inhibitor termed T. rubripes trypsin inhibitor 2 (TRTI-2) is a serine protease inhibitor and specifically inhibits trypsin with the inhibitory constant (Ki) of 8.6 × 10^–8 M. TRTI-2 is an acidic glycoprotein with a molecular mass of 63 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and deglycosylated to 46 kDa by treatment with glycopeptidase. Amino acid sequence and cDNA cloning analyses revealed that mature TRTI-2 comprises 421 amino acid residues with a theoretical molecular mass of 47,349.17 Da and isoelectric point (pI) of 5.47. A BLAST homology search revealed that TRTI-2 is homologous to hemopexin family protein and identical with fWap65-2, the warm temperature acclimation-related 65-kDa protein (Wap65) from T. rubripes. Wap65 shows functional diversification as a mediator of not only temperature acclimation but also inflammatory, antiinflammatory, and immune responses. The present study revealed a novel biochemical property of Wap65 as an antitrypsin, possibly to protect the functional molecule from damage by protease.

蛋白酶抑制剂广泛分布于鱼类的皮肤粘液中。河豚，河豚科，在鱼类中具有很高的抗胰蛋白酶活性。在这项研究中，我们从河豚Takifugu rubripes的皮肤粘液中分离并表征了蛋白酶抑制剂。名为T. rubripes胰蛋白酶抑制剂 2 (TRTI-2) 的抑制剂是一种丝氨酸蛋白酶抑制剂，可特异性抑制胰蛋白酶，抑制常数 ( Ki ) 为 8.6 × 10 ^–8 M. TRTI-2 是一种酸性糖蛋白，十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳显示分子量为 63 kDa，糖肽酶处理后去糖基化为 46 kDa。氨基酸序列和cDNA克隆分析表明成熟的TRTI-2包含421个氨基酸残基，理论分子量为47,349.17 Da，等电点(pI)为5.47。BLAST 同源性搜索显示，TRTI-2 与血红素结合蛋白家族蛋白同源，并且与 fWap65-2 相同，后者是来自T. rubripes的暖温适应相关 65-kDa 蛋白 (Wap65). Wap65 显示出功能多样化，不仅是温度适应的介质，而且是炎症、抗炎和免疫反应的介质。本研究揭示了 Wap65 作为抗胰蛋白酶的新生化特性，可能保护功能分子免受蛋白酶的损害。