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 specif icity (J.-Y. Li, K. Hoolfelder, K-S. Huang, and M. G. Low, J. Biol. Chem, 2 69, 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-phenan throline, could be completely restored with Zn2+ (and partially with Co2+), which indicates that Ca2+ can be removed from the protein without affectin g 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 muta nts in which the predicted Ca2+ binding sites were either deleted completel y or altered by single-residue substitution. All of the mutations showed su bstantial 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 ev en though it has no discernible role in the mature, secreted protein. (C) 1 999 Academic Press.