Cloning and expression of a cDNA encoding a Vorticella convallaria spasmin: an EF-Hand calcium-binding protein

The stalked, ciliated protozoan Vorticella convallaria possesses a highly c ontractile cytoskeleton consisting of spasmonemes and myonemes. The major c omponent of these contractile organelles is the calcium-binding protein(s) called spasmin. Cloning and characterization of spasmin would help elucidat e this contractile system. Therefore, enriched spasmoneme protein preparati ons from these contractile stalks were used to produce a monoclonal antibod y to spasmin. A monoclonal antibody, 1F5, was obtained that immunolocalized specifically to the spasmonemes and the myonemes and recognized a 20-kD ca lcium-binding protein in spasmoneme protein preparations. A putative spasmi n cDNA was obtained from a V. convallaria cDNA library and the derived amin o acid sequence of this cDNA revealed an acidic, 20-kD protein with calcium -binding helix-loop-helix domains. The physical properties of the putative spasmin were assessed by characterization of a recombinantly produced spasm in protein. The recombinant spasmin protein was shown to bind calcium using calcium gel-shift assays and was recognized by the anti-spasmin antibody. Therefore, a V. convallaria spasmin was cloned and shown to be a member of the EF-hand superfamily of calcium-binding proteins.