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.
Skip Nav Destination
Article navigation
Research Article|
March 01 2004
Effect of glycosylphosphatidylinositol (GPI)-phospholipase D overexpression on GPI metabolism
Karl J. MANN;
Karl J. MANN
*Department of Neuroscience, Mayo Clinic Jacksonville, 4500 San Pablo Road, Jacksonville, FL 32224, U.S.A.
Search for other works by this author on:
Matthew R. HEPWORTH;
Matthew R. HEPWORTH
*Department of Neuroscience, Mayo Clinic Jacksonville, 4500 San Pablo Road, Jacksonville, FL 32224, U.S.A.
Search for other works by this author on:
Nandita S. RAIKWAR;
Nandita S. RAIKWAR
†Departments of Medicine and Biochemistry and Molecular Biology, Indiana University School of Medicine and the Department of Veterans Affairs, IN 46202, U.S.A.
Search for other works by this author on:
Mark A. DEEG;
Mark A. DEEG
†Departments of Medicine and Biochemistry and Molecular Biology, Indiana University School of Medicine and the Department of Veterans Affairs, IN 46202, U.S.A.
Search for other works by this author on:
Daniel SEVLEVER
Daniel SEVLEVER
1
*Department of Neuroscience, Mayo Clinic Jacksonville, 4500 San Pablo Road, Jacksonville, FL 32224, U.S.A.
1To whom correspondence should be addressed (e-mail sevlever.daniel@mayo.edu).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
August 29 2003
Revision Received:
October 20 2003
Accepted:
November 11 2003
Accepted Manuscript online:
November 11 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 378 (2): 641–648.
Article history
Received:
August 29 2003
Revision Received:
October 20 2003
Accepted:
November 11 2003
Accepted Manuscript online:
November 11 2003
Citation
Karl J. MANN, Matthew R. HEPWORTH, Nandita S. RAIKWAR, Mark A. DEEG, Daniel SEVLEVER; Effect of glycosylphosphatidylinositol (GPI)-phospholipase D overexpression on GPI metabolism. Biochem J 1 March 2004; 378 (2): 641–648. doi: https://doi.org/10.1042/bj20031326
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Could not validate captcha. Please try again.