Recent studies have revealed that a growing number of cell surface proteins are attached to the membrane by covalent linkage to a glycosylphosphatidylinositol (GPI) (also called phosphatidylinositol-glycan (PI-G)) anchor. The physiological role played by this new class of membrane anchor is unknown, but one possibility is that it facilitates the release of molecules by specific phospholipases in vivo.
Several mammalian phospholipase activities which seem to be capable of removing the GPI anchors from proteins have been reported. These were originally attributed to the action of a phosphatidylinositol (PI)-specific phospholipase C since enzymes of this specificity are widely distributed in mammalian tissues. However, the physiological significance of such a process remained in question because almost all of the mammalian PI-specific phospholipase C's are believed to be intracellular in location whereas the GPI-anchored proteins are found on the cell surface. Subsequently, it was shown that inhibition of placental PI-specific phospholipase C activity does not affect GPI-anchor degrading activity, indicating that other enzymes are responsible for the release of GPI-anchored proteins. It was therefore suggested that this activity was due to a novel phospholipase D with specificity for the GPI-anchor. Recently, several groups have reported the presence of high levels of a GPI-specific phospholipase D (GPI-PLD), also called phosphatidylinositolglycan-specific phospholipase D in mammalian plasma and serum (6, 7, 8) Because of its extracellular location and specificity for GPI, this enzyme may be responsible for releasing GPI-anchored proteins from cell surfaces in vivo.
Secretion of proteins of interest from transfected eukaryotic cells permits the isolation and characterization of that protein free of the complex intracellular milieu and does so in the absence of detergents. Traditional methods for obtaining secretion have included attachment of secretory signal sequences to intracellular proteins or truncation of proteins to delete "retention signals" such as hydrophobic transmembrane domains. Many proteins have been successfully secreted to varying degrees using these strategies, but other proteins have remained obstinate for reasons that are usually not clear. An ideal method would be a dominant-acting post-translational modification that would specifically "tag" a protein for efficient secretion. While no such tag has yet been found, it appears that a naturally-occurring, dominant-acting modification has been identified that results in efficient cell-surface expression of the modified protein. Secretion of these proteins would then be realized by their specific release from the cell surface.
The modification referrred to is the post-translational transfer of a glycosylphosphatidylinositol (GPI) moiety to the COOH-terminal end of a protein (1,3,4,30-33). Numerous proteins in organisms ranging from trypanosomes to humans are anchored to the cell surface in this manner. The signal for GPI attachment is not completely defined by invariably includes a hydrophobic stretch of amino acids near the COOH-terminus of the initial translation product. These residues are proteolytically removed prior to en bloc attachment of the anchor (31). These GPI proteins are transported to the cell surface where the fatty acids of the GPI moiety serve to anchor the molecule to the outer leaflet of the lipid bilayer. The physiological role of this complex anchor is not clear, but proposals have included (1,3,4,33): 1) conferring on the protein high lateral mobility in the plasma membrane, and 2) generation of signal transducting molecules by a phospholipase-catalyzed hydrolysis of the GPI moiety.
Lisanti et al. (34) showed that expression of a chimeric gene encoding (at the 5' end) a protein that is normally secreted with (at the 3' end) a signal sequence; for GPI attachment resulted in the protein being localized at the cell surface. Furthermore, it can be demonstrated that GPI anchors act as dominant sorting signals by redirecting what are normally basolateral proteins to the apical side of polarized epithelial cells (1,3,4,33). While this activity can be exploited to obtain cell-surface expression of a protein of interest, it is also of interest to achieve secretion of proteins.
Molecules which are released in this way might be expected to contain a common COOH-terminus consisting of a hydrolyzed GPI anchor which is missing a phosphatidic acid moiety. The presence of a common structural epitope would facilitate the purification of these proteins. Antibodies specific for the phospholipase C cleaved GPI anchor, and anti-CRD reagents, have been previously described (33). Development of antibodies to a PLD cleaved GPI anchor would facilitate the purification of GPI PLD released GP anchored proteins.