| Popis: |
GPI8 is a Clan CD, family C13 cysteine protease and the catalytic core of the GPI: protein transamidase (GPIT) complex. GPI8 catalyses the addition of pre-formed glycosylphosphatidylinositol (GPI) anchors to the C-terminus region of GPI-anchored proteins. The GPIT complex has been well characterised in higher eukaryotes, and contains at least 4 components. In the parasitic protozoon Leishmania mexicana GPI8 is non-essential. Deltagpi8 mutants lack GPI-anchored GP63 from the cell surface. Site-directed mutagenesis of L. mexicana GPI8, followed by expression in the Δgpi8 cell line, identified the active site cysteine (C216) and histidine (H174) residues. The amino acid C94 was also identified as a functionally important residue. Mutation of the amino acid H65 had no detectable effect on GPI8 activity. Re-expression of GPI8C216G within WT cells had a dominant negative effect on the processing and trafficking of GP63 to the cell surface. This indicates that GPI8 is part of a larger complex within L. mexicana. Attempts to identify other components of the GPIT complex, by epitope tagging, or the production of a GPI-anchored GFP were unsuccessful. The Δgpi8 cell line was used to study the effect that a GPI-anchor deficiency has on the processing and trafficking of a GPI-protein. Non-GPI-anchored GP63 was secreted from the Δgpi8 cell line, and this secreted form was not processed in the same way as in WT cells. In Δgpi8 non-GPI-anchored GP63 was glycosylated and secreted without further processing from the cell with a ti/2 of 120 minutes. Loss in GPI anchoring did not result in the intracellular retention of GP63 as found in mammalian cells. N-glycans were shown to be important for the secretion of GP63 in the absence of a GPI anchor. In WT cells the majority of GP63 was rapidly glycosylated, GPI-anchored and trafficked to the surface with defined processing intermediates. WT cells secreted 2 isoforms of GP63 into the medium with different kinetics. The 65s isoform was not GPI-anchored, while the 63s isoform retained its anchor. It is suggested that anchored and unanchored GP63 are trafficked via 2 different pathways in Leishmania mexicana', a classical pathway whereby GP63 is N-glycosylated, GPI-anchored and then undergoes further modification during transport to the cell surface; or a direct secretion pathway whereby non-anchored GP63 is secreted from the cell without modification. |
