Structure and function of dedifferentiation factors with proteolytic and mitogenic activities in budding tunicates

我们以前的研究表明，在刚出芽的鳞片中，维甲酸是一种作用于间充质细胞的体轴诱导因子。然后，间充质细胞被认为可以分泌蛋白水解性因子，可以诱导多潜能细胞的去分化。本研究的目的是鉴定这些去分化因子的分子性质，并详细研究它们的功能。从穿心果菌落中分离纯化出40 kDa的氨基肽酶样多肽。对体外培养的多能细胞既有促生长作用，又有诱导肠特异性分化作用。根据N端的部分氨基酸序列和BrCN的内部裂解位点，扩增出PCR产物。以它们为探针，从穿心果cDNA文库中克隆了一个全长的cDNA。推导的氨基酸序列含有三叶生长因子基序和半胱氨酸蛋白酶基序，因此被称为被膜三叶因子(…更多TTF)。细菌蛋白表达系统构建的重组TTF对穿心果细胞株既无促生长活性，又无诱导分化活性。这一结果表明，TTF可能需要进行二次修饰，如糖链的添加才能发挥有丝分裂活性。重组TRAMP蛋白具有胰酶样蛋白分解活性。促进体外培养的多能上皮细胞生长。增殖中的细胞失去了胞浆颗粒等分化标记物，提示应发生去分化。具有与TRAMP相当的蛋白分解活性的正品胰酶不能诱导细胞增殖。这可能表明，细胞生长受到丝氨酸蛋白酶催化活性部位以外的未知结构域的调控。原位杂交和抗TRAMP免疫染色结果均表明，TAPAMP由发育芽中脱分化部位的间充质细胞表达。这些结果支持我们的工作假设，即在萌发的衣状物中，维甲酸诱导间充质细胞分泌蛋白水解多肽，从而调节多能细胞的生长和分化。较少

Our previous studies have shown that in the budding tunicate Polyandrocarpa misakiensis retinoic acid is a body axis-inducing factor which acts on mesenchymal cells. Then, the mesenchymal cells are thought to secrete proteolytic factors that can induce dedifferentiation of multipotent cells. The aim of this research project is to identify molecular nature of those dedifferentiation factors and to examine their functions in details.Amiriopeptidase-like polypeptide of 40 kDa has been purified from Polyandrocarpa colonies. It showed both activities of growth promotion and induction of gut-specific differentiation on cultured multipotent cells in vitro. From partial amino acid sequences at the N-terminus and an internal BrCN-cleaving site, PCR products were amplified. Using them as probes, a full length cDNA has been cloned from Polyandrocarpa cDNA library. Deduced amino acid sequence had trefoil growth factor motif and cysteine protease motif, thus referred to as tunicate trefoil factor ( … More tTF). Recombinant tTF raised by bacterial protein expression system showed neither growth-promoting activity nor differentiation-inducing activity on Polyandrocarpa cell lines. This result suggested that tTF might need secondary modifications such as the addition of sugar chains in order to exert the mitogenic activity.Recombinant TRAMP protein showed the trypsin-like proteolytic activity. It promoted in vitro cell growth of the multipotent epithelium. The proliferating, cells lost differentiation markers such as cytoplasmic granules, suggesting that dedifferentiation should occur. Authentic trypsin with the proteolytic activity comparable to TRAMP dud not induce cell proliferation. This may indicate that cell growth is regulated by unknown domains other than catalytic active sites of serine proteases. Results of both in situ hybridization and anti-TRAMP immunostaining have shown that TPAMP is expressed by mesenchymal cells located at the site where dedifferentiation occurs in developing buds. These results support our working hypothesis that in budding tunicates retinoic acid induces mesenchymal cells to secrete proteolytic polypeptides that can regulate growth and differentiation of multipotent cells. Less