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

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 GPIanchored 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 GPIanchored 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.

A Proteomic Analysis of the Plasma Glycoproteins of a MCF-7 Mouse Xenograft: A Model System for the Detection of Tumor Markers

“…A related protein found in this study is GPI-PLD which cleaves the GPI anchor sequence attached to many cell surface proteins including receptors. 57 GPI-PLD was downregulated in the estrogen and combined treatment groups, which is consistent with reports that this protein was decreased during inflammation 58 and which can be related to tumor growth.…”

Shedding and uptake of gangliosides and glycosylphosphatidylinositol-anchored proteins

“…Initial reports indicated that GPI-PLD was active against GPI-anchored proteins only in the presence of detergent, and was not able to cleave the anchors of proteins in a native membrane context [218]. Overexpression experiments indicated that it is active in endoplasmic reticulum during GPI synthesis, but also in lipid rafts [219]. Lipid fluidity and packing are the most important modulators of bacterial phospholipase ability to cleave GPI anchors [220] and modulation of membrane lipids were reported to affect GPI-PLD activity in vitro [221], so it is quite possible that mammalian GPI-PLD also requires particular membrane composition for activity.…”

Alkaline Phosphatase Inhibitors Attenuate Renovascular Responses to Norepinephrine

“…TNAP is a GPI-anchored ecto-enzyme 22 , and in the secretory pathway GPI-PLD cleaves the GPI anchor from TNAP 37–41 , and TNAP becomes a secreted, soluble enzyme. GPI-PLD activity is inhibited by protein kinase A (PKA)-mediated phosphorylation of threonine 286 42 .…”