Posttranslational modification of glycosylphosphatidylinositol (GPI)-specific phospholipase D and its activity in cleavage of GPI anchors.

Human glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was exogenously expressed in mammalian CHO cells and in insect H5 cells. GPI-PLD was initially synthesized as a 105-kDa form and then secreted as a mature 115-kDa form from the CHO cells, whereas it was secreted as an immature 98-kDa form from the H5 cells. The difference of the molecular forms was caused by its oligosaccharide processing in the two cell lines. These forms showed a different reactivity to anti-C-terminal peptide of GPI-PLD; the 105-kDa and 98-kDa forms were directly recognized by the antibodies, whereas the 115-kDa form was immunoreactive only after being denatured. In an in vitro assay, the 98-kDa form but not the 115-kDa form was able to release a significant amount of GPI-anchored proteins from intact membranes, although the two forms had the same GPI-anchor cleavage activity in the presence of detergents. In addition, a GPI-anchored protein, when coexpressed in CHO cells, was intracellularly cleaved by GPI-PLD in the secretory pathway. Taken together, these results suggest that GPI-PLD undergoes a conformational change by posttranslational modification, which affects its immunoreactive and enzymatic properties.