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+)](0.5) 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.
Granulocyte Research Laboratory, Department of Hematology, Rigshospitalet, 2100 Copenhagen, Denmark.
K
K
Lollike
A H
AH
Johnsen
I
I
Durussel
N
N
Borregaard
J A
JA
Cox
D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D009504:Neutrophils / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding
D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; C503121:GCA protein, human; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; C026742:plastin; D002118:Calcium