Expression of bloodstream variant surface glycoproteins in procyclic stage Trypanosoma brucei: role of GPI anchors in secretion.
Using transformed procyclic trypanosomes, the synthesis, intracellular transport and secretion of wild-type and mutant variant surface glycoprotein (VSG) is characterized. We find no impediment to the expression of this bloodstream stage protein in insect stage cells. VSG receives a procyclic-type phosphatidylinositol-specific phospholipase C-resistant glycosyl phosphatidylinositol (GPI) anchor, dimerizes and is N-glycosylated. It is transported to the plasma membrane with rapid kinetics (t(1/2) approximately 1 h) and then released by a cell surface zinc-dependent metalloendoprotease activity, a possible homolog of leishmanial gp63. Deletion of the C-terminal GPI addition signal generates a soluble form of VSG that is exported with greatly reduced kinetics (t(1/2) approximately 5 h). Fusion of the procyclic acidic repetitive protein (PARP) GPI anchor signal to the C-terminus of the truncated VSG reporter restores both GPI addition and transport competence, suggesting that GPI anchors play a critical role in the folding and/or forward transport of newly synthesized VSG. The VSG-PARP fusion is also processed near the C-terminus by events that do not involve N-linked oligosaccharides and which are consistent with GPI side chain modification. This unexpected result suggests that GPI processing may be influenced by adjacent peptide sequence or conformation.
Bangs
J D
JD
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, 53706, USA. bangs@macc.wisc.edu
Ransom
D M
DM
McDowell
M A
MA
Brouch
E M
EM
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D017930:Genes, Reporter / Q000235:genetics; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D006596:Hexosaminidases / Q000378:metabolism; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D019308:Palmitic Acid / Q000378:metabolism; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; C053277:procyclic acidic repetitive protein, Trypanosoma; D019308:Palmitic Acid; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D006596:Hexosaminidases; D008666:Metalloendopeptidases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D015032:Zinc