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 fluid s. 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 e ncoding a signal peptide-deficient and a GPI-anchored form of GPI-PLD and transiently transfected these constructs into COS-1 cells. The sig nal peptide-deficient form of GPI-PLD was expressed as a 90-kDa protei n that was catalytically active and was localized intracellularly. Cel ls transfected with cDNA encoding the GPI-anchored form of GPI-PLD exp ressed a catalytically active enzyme of 100 kDa that could be labelled with [H-3]ethanolamine demonstrating its modification by a GPI struct ure. Expression of the GPI-anchored form of GPI-PLD resulted in the re lease of endogenous GPI-anchored alkaline phosphatase from COS-I cells , whereas expression of the intracellular form of GPI-PLD had no effec t on membrane attachment of endogenous alkaline phosphatase. Similarly , in cells cotransfected with GPI-anchored placental alkaline phosphat ase (FLAP) and the GPI-anchored form of GPI-PLD, PLAP was released int o the cell culture supernatant while expression of the signal peptide- deficient form of GPI-PLD did not affect the amount of cell-associated FLAP. (C) 1997 Elsevier Science B.V.