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.
Li
J Y
JY
College of Physicians and Surgeons, Columbia University, New York, New York, 10032, USA.
Low
M G
MG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D002417:Cattle; D004789:Enzyme Activation / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D016023:Integrins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D010739:Phospholipase D / Q000378:metabolism*; D014162:Transfection; D015032:Zinc / Q000502:physiology
D002135:Calcium-Binding Proteins; D016023:Integrins; D010446:Peptide Fragments; D010739:Phospholipase D; C065017:glycoprotein phospholipase D; D015032:Zinc; D002118:Calcium