Biochemical characterization of the penta-EF-hand protein grancalcin and identification of L-plastin as a binding partner.

Grancalcin is a recently described Ca(2+)-binding protein especially abundant in human neutrophils. Grancalcin belongs to the penta-EF-hand subfamily of EF-hand proteins, which also comprises calpain, sorcin, peflin, and ALG-2. Penta-EF-hand members are typified by two novel types of EF-hands: one that binds Ca(2+) although it has an unusual Ca(2+) coordination loop and one that does not bind Ca(2+) but is directly involved in homodimerization. We have developed a novel method for purification of native grancalcin and found that the N terminus of wild-type grancalcin is acetylated. This posttranslational modification does not affect the secondary structure or conformation of the protein. We found that both native and recombinant grancalcin always exists as a homodimer, regardless of the Ca(2+) load. Flow dialysis showed that recombinant grancalcin binds two Ca(2+) per subunit with positive cooperativity and moderate affinity (Ca(2+) of 25 and 83 microm in the presence and absence of octyl glycoside, respectively) and that the sites are of the Ca(2+)-specific type. Furthermore, we showed, by several independent methods, that grancalcin undergoes important conformational changes upon binding of Ca(2+) and subsequently exposes hydrophobic amino acid residues, which direct the protein to hydrophobic surfaces. By affinity chromatography of solubilized human neutrophils on immobilized grancalcin, L-plastin, a leukocyte-specific actin-bundling protein, was found to interact with grancalcin in a negative Ca(2+)-dependent manner. This was substantiated by co-immunoprecipitation of grancalcin by anti-L-plastin antibodies and vice versa.