Glycophospholipid membrane anchor attachment. Molecular analysis of the cleavage/attachment site

The COOH terminus of decay accelerating factor (DAF) contains a signal that directs attachment of a glycophosphatidylinositol (GPI) membrane anchor in a process involving proteolytic removal of 17-31 COOH-terminal residues. Previous work suggested that two elements are required for anchor addition, a COOH-terminal hydrophobic domain (the GPI signal) and an element located NH2-terminal to it, postulated to be the cleavage/attachment site. Using [3H]ethanolamine (a component of the anchor) to tag the COOH terminus, we isolated and sequenced a COOH-terminal tryptic peptide, thereby identifying Ser-319 as the COOH-terminal residue attached to the GPI anchor. This indicates that a 28-residue peptide is removed during processing and localizes the cleavage/attachment site precisely to the region previously shown to be required for anchor attachment (between 10 and 20 residues NH2-terminal to the hydrophobic domain). Since DAF contains multiple cryptic cleavage/attachment sites, we used a GPI-linked human growth hormone-DAF fusion to study the structural requirements for cleavage/attachment. Our results show that while sequences immediately NH2-terminal to the attachment site are not required for anchor addition, deletion of Ser-319 abolishes both anchor attachment and transport to the cell surface. Systematic replacement of the attachment site serine with all possible amino acids indicated that alanine, aspartate, asparagine, glycine, or serine efficiently support GPI anchor attachment while valine and glutamate are partially effective. All other substitutions including cysteine (permitted at the attachment site in other GPI-anchored proteins) abolish both GPI anchor attachment and transport to the cell surface, resulting in accumulation of uncleaved fusion protein in internal compartments (endoplasmic reticulum and Golgi). These results support the general rule that the residue at the cleavage/attachment site must be small. Further, addition of a GPI anchor appears to be necessary for transport to the cell surface in transfected COS cells.