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J. Biochem, 2004, Vol. 135, No. 1 71-78
© 2004 The Japanese Biochemical Society

BIOTECHNOLOGY

Phospholipids and Their Derivatives as Mitogen and Motogen of Budding Tunicates

Kazuya Arai1, Satoshi Yoshida2, Masanao Nakatani1, Shigeki Fujiwara1, Toshitsugu Yubisui1,3 and Kazuo Kawamura*,1

1 Laboratory of Cellular and Molecular Biotechnology, Faculty of Science, Kochi University, Kochi 780-8520; 2 Department of Bimolecular Science, Faculty of Engineering, Gifu University, Gifu 501-1193; and 3 Department of Biological Chemistry, Faculty of Science, Okayama University of Science, Okayama 700-0005

In order to discover novel invertebrate cytokines from the budding tunicate, Polyandrocarpa misakiensis, we treated the water-insoluble fraction of tunicate homogenates with trypsin. The extracts showed remarkable activities to promote the growth and motility of tunicate cells. The activities were heat-stable and proteinase K-resistant. After anion exchange chromatography, the activities were eluted with detergents such as 0.1% deoxycholic acid. The Fourier transform infrared spectrum indicated large amounts of fatty acids and phospholipids instead of polypeptides in the extracts. Consistently, the activities were extractable with organic solvents such as chloroform. Long chains of n-3 polyunsaturated free fatty acids (FFA), phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) were the major components in the lipid-soluble fraction. A cDNA for FFA-releasing enzyme phospholipase A2 (PLA2) was cloned. The expression of this gene could be seen in epidermal cells during budding. The recombinant protein, as in the case of the authentic PLA2, preferred PC and PE as substrates, followed by PS and PI. The resultant FFAs only promoted cell growth, while the remaining lysophospholipids stimulated cell motility. The former contained unsaturated fatty acids (C18:1, C20:5, and C22:6) while the latter did not, suggesting that unsaturated fatty acids are responsible for mitogenic activity in tunicate cells. These results show for the first time that phospholipids and their derivatives are bio-mediators promoting cell growth and cell motility in invertebrates.

* To whom correspondence should be addressed. Fax: +81-88-844-8313, E-mail: kazuk{at}cc.kochi-u.ac.jp


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