Functional characteristics and the complete primary structure of ascidian gelsolin

Abstract

The body wall of the ascidian is composed of unusual multi-nucleated smooth muscle cells enriched with thin actin filaments containing troponin–tropomyosin which run along the longitudinal cell axis without being organized into striated structures. We purified an actin-binding protein of 80 kDa, tentatively termed 80K protein, from the body wall muscle of ascidian, Halocynthia roretzi, and characterized the functional properties and molecular structures. In the presence of Ca 2+, the 80K protein accelerated the initial phase of actin polymerization, namely the nucleation process, decreased the level of polymerization at the steady state, caused marked reduction in viscosity of an F-actin solution, and fragmented F-actin filaments, while in the absence of Ca 2+, it remained associated with F-actin without severing the filaments. The interaction of the 80K protein with actin was inhibited by phosphatidylinositol 4,5-bisphosphate (PIP 2). When actin was polymerized in the presence of acrosome actin bundles from horseshoe crab sperm, the 80K protein inhibited the growth of actin filaments at the barbed end but not at the pointed end, indicating that the 80K protein functions as a barbed-end capping protein. In order to characterize the molecular structure of the 80K protein, cDNAs encoding this protein were isolated from the λgt11 cDNA library of the ascidian muscle by using a monoclonal antibody (AS23) specific for this protein and the entire sequence was determined. The deduced peptide sequence showed about 44% homology in amino acid residues with the human gelsolin sequence, and in addition, 6 repeating segments were observed in the sequence of the 80K protein as has been described in the gelsolin sequence. These results indicate strongly that the 80K protein belongs to the gelsolin family.