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

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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