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.
Caras
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IW
Genentech, Inc., South San Francisco, CA 94080.
Moran
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D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis* / Q000737:chemistry; D008322:Mammals; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology / Q000502:physiology; D014346:Trypanosoma brucei brucei / Q000378:metabolism*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000737:chemistry / Q000378:metabolism*
D000941:Antigens; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma