Biochemical Characterization of Copine:  A Ubiquitous Ca2+-Dependent, Phospholipid-Binding Protein

The copines are a novel group of Ca2+-dependent, phospholipid-binding proteins first isolated from Paramecium tetraurelia [Creutz, C. E., et al. (1998) J. Biol. Chem. 273, 1393−1402] and found in a wide range of organisms, from plants to humans. They have a Ca2+ and phospholipid-binding domain consisting of two C2 domains and a core domain in the C-terminal portion that is homologous to the A domain found in certain integrins. We provide here the first description of the properties and distribution of a native mammalian copine, copine I. This protein is expressed in all major adult rat organs as demonstrated by probing Western blots of rat organ homogenates with anticopine antibodies. The highest levels of copine are found in the spleen. A protocol for purifying copine to homogeneity from bovine spleen is described. Purified native copine is a 58 kDa monomer that exhibits Ca2+ self-association to form higher-order multimers, and Ca2+-dependent, phospholipid binding activity with preference for negatively charged phospholipids over neutral phospholipids and selectivity for Ca2+ over Mg2+. Half-maximal association with vesicles enriched in phosphatidylserine occurs at Ca2+ concentrations between 1 and 10 μM. Copine I exhibits Mn2+ binding activity that is strongly competed by Mg2+ and partially competed by Ca2+, suggesting that the copine I A domain may be a functional MIDAS metal binding site similar to that found in integrins [Lee, J. O., et al. (1995) Cell 80, 631−638]. Roles for copine in binding membranes and target proteins or small molecules are discussed.