Expression of intracellular and GPI-anchored forms of GPI-specific phospholipase D in COS-1 cells.
Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is a secretory protein present in high amounts in mammalian body fluids. Its cDNA has been isolated and encodes a signal peptide of 23 amino acids and the mature protein of 816 amino acids. We generated cDNAs encoding a signal peptide-deficient and a GPI-anchored form of GPI-PLD and transiently transfected these constructs into COS-1 cells. The signal peptide-deficient form of GPI-PLD was expressed as a 90-kDa protein that was catalytically active and was localized intracellularly. Cells transfected with cDNA encoding the GPI-anchored form of GPI-PLD expressed a catalytically active enzyme of 100 kDa that could be labelled with [3H]ethanolamine demonstrating its modification by a GPI structure. Expression of the GPI-anchored form of GPI-PLD resulted in the release of endogenous GPI-anchored alkaline phosphatase from COS-1 cells, whereas expression of the intracellular form of GPI-PLD had no effect on membrane attachment of endogenous alkaline phosphatase. Similarly, in cells cotransfected with GPI-anchored placental alkaline phosphatase (PLAP) and the GPI-anchored form of GPI-PLD, PLAP was released into the cell culture supernatant while expression of the signal peptide-deficient form of GPI-PLD did not affect the amount of cell-associated PLAP.
Küng
M
M
Institute of Biochemistry and Molecular Biology, University of Bern, Switzerland.
Bütikofer
P
P
Brodbeck
U
U
Stadelmann
B
B
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D010739:Phospholipase D / Q000096:biosynthesis*; D014162:Transfection; D010738:Type C Phospholipases / Q000494:pharmacology
D017261:Glycosylphosphatidylinositols; D000469:Alkaline Phosphatase; D010738:Type C Phospholipases; D010739:Phospholipase D