Numerous cell surface proteins are anchored to the cell membrane by covalent linkage to a complex lipid moiety, glycosylphosphatidylinositol (GPI). The proteins which utilize the GPI anchor are functionally and evolutionarily diverse, and have been identified on enzymes, adhesion molecules, receptors, etc. in all eukaryotic cell types examined so far. It has been proposed that the presence of a GPI anchor on a particular protein facilitates specific interactions with other molecules at the cell surface. It also provides a means for rapid release from the membrane as a result of degradation by specific phospholipases. However, the reason why such a complex membrane protein anchoring mechanism should be so widely distributed in nature remains obscure. In mammalian cells, GPI anchors can become acylated on the inositol ring which blocks their degradation by phospholipase C, but not by phospholipase D. Although inositol acylation has a profound effect on the biochemical properties of the GPI-anchored proteins, little is known about the biosynthesis of this modification or what effect it has on the function of GPI-anchored proteins. In this proposed research, the mechanism of biosynthesis and functional significance of inositol acylation will be investigated in human tumor cell lines. The subcellular location of the inositol acylation biosynthetic process will be determined by pulse-chase studies. In vitro enzyme assays and mutant cell lines will be developed to facilitate studies of the inositol-acylation process at the molecular level. The function of inositol acylation will be determined by studying its effect on several important properties of GPI-anchored proteins: (I) specific interactions with cell surface lipids and non-receptor tyrosine kinases, (ii) accessibility in the cell surface to degradation by plasma phospholipase D, and (iii) participation in T lymphocyte activation. The results of these studies will provide valuable insights into the biochemistry and cell biology of an increasingly important mechanism of membrane anchoring. GPI-anchored proteins have many important functions on the surface of human cells and inositol acylation is likely to have a major influence on their ability to carry-out those functions correctly. The results of these studies will therefore have significant implications for understanding the molecular basis of human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035873-12
Application #
2734535
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1987-07-01
Project End
2000-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
12
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Physiology
Type
Schools of Medicine
DUNS #
167204994
City
New York
State
NY
Country
United States
Zip Code
10032
Du, Xiaohan; Cai, Jiewei; Zhou, Jian-zhong et al. (2002) Tolerance of glycosylphosphatidylinositol (GPI)-specific phospholipase D overexpression by Chinese hamster ovary cell mutants with aberrant GPI biosynthesis. Biochem J 361:113-8
Du, X; Low, M G (2001) Down-regulation of glycosylphosphatidylinositol-specific phospholipase D induced by lipopolysaccharide and oxidative stress in the murine monocyte- macrophage cell line RAW 264.7. Infect Immun 69:3214-23
Low, M G; Stutz, P (1999) Inhibition of the plasma glycosylphosphatidylinositol-specific phospholipase D by synthetic analogs of lipid A and phosphatidic acid. Arch Biochem Biophys 371:332-9
Li, J Y; Low, M G (1999) Studies of the role of the integrin EF-hand, Ca2+-binding sites in glycosylphosphatidylinositol-specific phospholipase D: reduced expression following mutagenesis of residues predicted to bind Ca2+. Arch Biochem Biophys 361:142-8
Xie, M; Low, M G (1995) Streptolysin-O induces release of glycosylphosphatidylinositol-anchored alkaline phosphatase from ROS cells by vesiculation independently of phospholipase action. Biochem J 305 ( Pt 2):529-37
Xie, M; Low, M G (1994) Identification and characterization of an ecto-(lyso)phosphatidic acid phosphatase in PAM212 keratinocytes. Arch Biochem Biophys 312:254-9
Misra, K B; Kim, K C; Cho, S et al. (1994) Purification and characterization of adipocyte heparan sulfate proteoglycans with affinity for lipoprotein lipase. J Biol Chem 269:23838-44
Xie, M; Low, M G (1994) Expression and secretion of glycosylphosphatidylinositol-specific phospholipase D by myeloid cell lines. Biochem J 297 ( Pt 3):547-54
Sillence, D J; Low, M G (1994) Hydrolysis of cell surface inositol phospholipid leads to the delayed stimulation of phosphatidylinositol synthesis in bovine aortic endothelial cells. Biochim Biophys Acta 1224:247-54
Wong, Y W; Low, M G (1994) Biosynthesis of glycosylphosphatidylinositol-anchored human placental alkaline phosphatase: evidence for a phospholipase C-sensitive precursor and its post-attachment conversion into a phospholipase C-resistant form. Biochem J 301 ( Pt 1):205-9

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