The requirements for GPI-attachment are similar but not identical in mammalian cells and parasitic protozoa.

To test whether the requirements for GPI-attachment are the same in mammalian cells and parasitic protozoa, we expressed the GPI-linked variant surface glycoprotein (VSG) of Trypanosoma brucei (T. brucei) in COS cells. Although large amounts of VSG were produced, only a small fraction became GPI-linked. This impaired processing is not due to the VSG ectodomain since replacement of the VSG GPI-signal with that of decay accelerating factor (DAF) produced GPI-linked VSG. Further, whereas fusion of the DAF GPI-signal to the COOH-terminus of human growth hormone (hGH) produces GPI-linked hGH, an analogous fusion using the VSG GPI-signal does not, indicating that the VSG GPI-signal functions poorly in mammalian cells. By constructing chimeric VSG-DAF GPI-signals and fusing them to the COOH-terminus of hGH, we show that of the two critical elements that comprise the GPI-signal--the cleavage/attachment site and the hydrophobic domain--the former is responsible for the impaired activity of the VSG GPI-signal in COS cells. To confirm this, we show that the VSG GPI-signal can be converted to a viable signal for mammalian cells by altering the amino acid configuration at the cleavage/attachment site. We also show that when fused to hGH, the putative GPI-signal from the malaria circumsporozoite (CS) protein produces low levels of GPI-anchored hGH, suggesting that the CS protein is indeed GPI-linked, but that the CS protein GPI-signal, like the VSG-signal, functions poorly in COS cells.