中国科学院昆明动物研究所机构知识库

三叶因子是由几个或者一个三叶因子结构域构成的蛋白多肽，三叶因子广泛存在于哺乳类至两栖类中，而且具有较高的保守性。目前研究较多的是来源于哺乳类的三叶因子，其主要表达与胃肠道，比较明确的功能是粘膜修复。对于其发挥作用的机制也有一些探究，但是目前三叶因子的作用受体一直悬而未决；相较于哺乳类来源的三叶因子，两栖类来源的三叶因子在皮肤也大量分泌，三叶因子种类也较多，但两栖类来源的三叶因子的功能研究很少。通过对云南大蹼铃蟾皮肤分泌物的重新分离，我们得到了一类新的三叶因子，将其命名为BmTFF3，并且进一步的质谱鉴定表明BmTFF3是复合物βg-CAT的β亚基。βg-CAT是一类新型三叶因子和aerolysin类似蛋白复合而形成得的分泌型的孔道形成蛋白。BmTFF3通过促进小鼠皮肤伤口的重新上皮化以及胶原排列而帮助小鼠皮肤伤口愈合。另外一方面，在DSS诱导的结肠炎小鼠模型中，腹腔给药（BmTFF3）能够改善结肠炎小鼠体重和结肠长度等方面的症状。这些研究表明BmTFF3发挥着三叶因子传统的保护功能，也提示着在两栖类的皮肤也有可能具有保护作用。BmTFF3与其他三叶因子的不同之处在于能够形成孔道形成蛋白复合物（βg-CAT），研究BmTFF3在其中的作用有助于发现三叶因子的更多功能。相比较于复合物βg-CAT，BmTFF3不能引起红细胞溶血；与类似分子BmTFF2相比，BmTFF3不具有血小板聚集活性。在免疫功能方面，高浓度的BmTFF3能够促进THP-1释放成熟的IL-1β，与复合物βg-CAT相似，但是所需浓度高达1 μM。BmTFF3抗体中和实验结果显示：BmTFF3抗体通过抑制复合物βg-CAT结合细胞膜并进一步阻止其寡聚化进而完全抑制复合物βg-CAT的生物学活性，体外结合实验发现，BmTFF3可以结合复合物βg-CAT的作用分子，表明BmTFF3在复合物中主要发挥结合受体作用。其次，在37℃的条件下，BmTFF3能够稳定复合物βg-CAT，而类似分子BmTFF2不具有稳定作用。本研究对两栖类来源的三叶因子具有更深的认识，也提示了三叶因子的作用受体。

Trefoil factors are widely present in mammals to amphibians and are highly conserved, which consisting of several or one trefoil factor domain. In mammals, trefoil factors have the role of mucosal repair. Differently, the amphibian-derived trefoil factor are more kinds of structures and is secreted in the skin other than gastrointestinal tract. Howerve, its mechanism is still unkown, as well as its receptors. As far as the function of amphibian-derived trefoil factors is less researched.We discovered a new trefoil factor, named BmTFF3, and further MS identification indicated that BmTFF3 is the β subunit of the complex βg-CAT. βg-CAT is is the first reported complex of secreted trefoil factor and aerolysin-like protein. BmTFF3 was found to have good wound healing ability in mouse skin by promoted the re-epithelization and collagen arrangement of mouse skin wounds. On the other hand, in mouse models of colitis induced by DSS, intraperitoneal administration (BmTFF3) could improve the symptoms of colitis. These studies suggest that BmTFF3 plays a traditional protection function, as well as amphibious skin.The difference between BmTFF3 and other trefoil factors is the ability to form a pore forming protein complex (βg-CAT), and studying the role of BmTFF3 of the complex helps to discover more functions of the trefoil factor. Compared to the complex βg-CAT show high activity of hemolysis to ammalian erythrocyte, BmTFF3 was not sensitive to erythrocyte; compared to the similar molecule BmTFF2, BmTFF3 hadn’t platelet aggregation activity. In terms of immune function, BmTFF3 could promote the release of mature IL-1β from THP-1 on high concentrations( 1μM). The antibody ( anti-BmTFF3) could completely inhibit the biological activity of complex βg-CAT by suppressd compelx’s combination with the cell membrane and its subsequent oligomerization. Second, BmTFF3 could bind sulfatide, one of the interacting molecular of complex βg-CAT. Then, BmTFF3 could stabilize the complex βg-CAT in vitro, while the similar molecule BmTFF2 had not the same effect. BmTFF3 not only has the traditional protective effect of the trifoil factor, but also bind the receptor and guide the complex βg-CAT to bind the cells, which leads to the further oligomerization and the biological effect. This study has a deeper understanding of amphibians-derived trefoil factors and suggests the role of trefoil factor receptors.