Effect of glycosylphosphatidylinositol (GPI)-phospholipase D overexpression on GPI metabolism

Karl J. Mann, Matthew R. Hepworth, Nandita S. Raikwar, Mark A. Deeg, Daniel Sevlever

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

GPI-PLD [glycosylphosphatidylinositol (GPI)-specific phospholipase D (PLD)] is a secreted mammalian enzyme that specifically cleaves GPI-anchored proteins. In addition, the enzyme has been shown to cleave GPI anchor intermediates in cell lysates. The biosynthesis of the GPI anchor is well characterized; however, the mechanisms by which the levels of GPI anchor intermediates are regulated are still unknown. To investigate whether GPI-PLD plays a role in this regulation, we isolated stable HeLa cells overexpressing the enzyme. GPI-PLD-HeLa (GPI-PLD-transfected HeLa) cells showed a 3-fold increase in intracellular GPI-PLD activity and drastically decreased the levels of GPI-anchored proteins when compared with untransfected HeLa controls. Intracellular cleavage of GPI-anchored proteins has been suggested to occur early in the secretory pathway and, in agreement with this proposal, GPI-PLD activity in GPI-PLD-HeLa cells was detected not only in the endoplasmic reticulum and Golgi apparatus, but also in the plasma membrane. The enzyme was also active in lipid rafts, membrane microdomains in which GPI-anchored proteins and GPI anchor intermediates are concentrated, indicating that intracellular GPI-PLD cleavage may also occur in this compartment. Pulse-chase paradigms revealed the turnover rate of the last intermediate of the GPI anchor pathway in GPI-PLD-HeLa cells to be accelerated compared with the controls. Furthermore, 1,10-phenanthroline, a GPI-PLD inhibitor, reversed this effect. Our studies demonstrated that GPI-PLD can cleave not only GPI-anchored proteins, but also GPI anchor intermediates intracellularly. This observation opens the possibility that GPI-PLD can influence the steady-state levels of GPI-anchored proteins by hydrolysing the anchor before and after its attachment to proteins.

Original languageEnglish (US)
Pages (from-to)641-648
Number of pages8
JournalBiochemical Journal
Volume378
Issue number2
DOIs
StatePublished - Mar 1 2004

Keywords

  • Glycosylphosphatidylinositol (GPI)
  • Glycosylphosphatidylinositol-anchored protein
  • Glycosylphosphatidylinositol-specific phospholipase D
  • HeLa cell
  • Lipid raft

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Effect of glycosylphosphatidylinositol (GPI)-phospholipase D overexpression on GPI metabolism'. Together they form a unique fingerprint.

Cite this