Studies of the Role of the Integrin EF-Hand, Ca2+-Binding Sites in Glycosylphosphatidylinositol-Specific Phospholipase D: Reduced Expression Following Mutagenesis of Residues Predicted to Bind Ca2+

Previous studies of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) have demonstrated that GPI-PLD can bind Ca2+ions with high specificity (J.-Y. Li, K. Hollfelder, K.-S. Huang, and M. G. Low,J. Biol. Chem.269, 28063–28971, 1994). In this study the functional role of the bound Ca2+ions was evaluated. The enzymatic activity of purified GPI-PLD, which was depleted of divalent cations by pretreatment with EDTA, EGTA, or 1,10-phenanthroline, could be completely restored with Zn2+(and partially with Co2+), which indicates that Ca2+can be removed from the protein without affecting its enzymatic activity. This result suggested that Ca2+bound to GPI-PLD has a structural or regulatory role but is not required for GPI hydrolysis. To evaluate these possibilities we transfected COS cells with GPI-PLD mutants in which the predicted Ca2+-binding sites were either deleted completely or altered by single-residue substitution. All of the mutations showed substantial reductions in the amount of GPI-PLD secreted into the medium (0–6% of wild type). The data indicate that bound Ca2+plays an important role in the initial folding, intracellular transport, or secretion of GPI-PLD even though it has no discernible role in the mature, secreted protein.