Mammalian glycophosphatidylinositol anchor transfer to proteins and posttransfer deacylation

Abstract

The glycophosphatidylinositol (GPI) anchors of proteins expressed on human erythrocytes and nucleated cells differ with respect to acylation of an inositol hydroxyl group, a structural feature that modulates their cleavability by PI-specific phospholipase C (PI-PLC). To determine how this GPI anchor modification is regulated, the precursor and protein-associated GPIs in two K562 cell transfectants (ATCC and .48) exhibiting alternatively PI-PLC-sensitive and resistant surface proteins were analyzed and the temporal relationship between GPI protein transfer and acquisition of PI-PLC sensitivity was determined. Nondenaturing PAGE analyses demonstrated that, whereas in .48 transfectants the GPI anchors in decay accelerating factor (DAF) and placental alkaline phosphatase (PLAP) were >95% acylated, in ATCC transfectants, they were 60 and 33% unsubstituted, respectively. In contrast, TLC analyses revealed that putative GPI donors in the two lines were identical and were >95% acylated. Studies of de novo DAF biosynthesis in HeLa cells bearing proteins with >90% unacylated anchors showed that within 5 min at 37°C (or at 18°C, which does not permit endoplasmic reticilum exit), >50% of the anchor in nascent 44-kDa proDAF protein exhibited PI-PLC sensitivity. In vitro analyses of the microsomal processing of miniPLAP, a truncated PLAP reporter protein, demonstrated that the anchor donor initially transferred to prominiPLAP was acylated and then progressively was deacylated. These findings indicate that (i) the anchor moiety that initially transfers to nascent proteins is acylated, (ii) inositol acylation in mature surface proteins is regulated via posttransfer deacylation, which in general is cell-specific but also can be proteindependent, and (iii) deacylation occurs in the endoplasmic reticulum immediately after GPI transfer.

Inositol Deacylation by Bst1p Is Required for the Quality Control of Glycosylphosphatidylinositol-anchored Proteins

“…In mammalian cells, a mammalian model GPI-anchored protein, DAF, was 56% deacylated in the ER within as little as 5 min (Chen et al, 1998). Inositol deacylation of GPI is important for the efficient transport of GPI-anchored proteins from the ER to the Golgi (Tanaka et al, 2004).…”

“…The amounts of GPI-anchored proteins are greatly decreased in gwt1 mutant cells, indicating that this acylation is critical for the attachment of GPI to proteins (Umemura et al, 2003). Once the GPI anchor is attached to a protein, the inositol is usually deacylated in the ER (Figure 1) (Chen et al, 1998). Recently, mammalian PGAP1 and the yeast orthologue Bst1p were identified as GPI inositol deacylases (Tanaka et al, 2004).…”

Male-specific Modification of Human CD52

“…3 This difference between the two species counterparts might originate from differences in the GPI anchor biosynthesis of the male genital tract epithelium. Intracellular GPI donors contain predominantly acylated inositol, and inositol acylation of mature cell surface proteins is regulated via post-transfer deacylation which in general seems to be cell type-specific (32).…”

Inositol Deacylation of Glycosylphosphatidylinositol-anchored Proteins Is Mediated by Mammalian PGAP1 and Yeast Bst1p

“…It is very likely that the acyl group is required for a later step in the pathway when "bridging" ethanolamine phosphate, which links GPI to the protein, is added to the third mannose to generate mature GPI. Soon after the attachment of GPI to proteins, the inositol is usually deacylated in the ER and becomes sensitive to PI-PLC (6). Human erythrocytes represent an exception, in which the inositol remains acylated, and all the GPI-APs are resistant to PI-PLC (7)(8)(9).…”