Persistence of Trypanosoma brucei as early procyclic forms and social motility are dependent on glycosylphosphatidylinositol transamidase.

Glycosylphosphatidylinositol (GPI)-linked molecules are surface-exposed membrane components that influence the infectivity, virulence and transmission of many eukaryotic pathogens. Procyclic (insect midgut) forms of Trypanosoma brucei do not require GPI-anchored proteins for growth in suspension culture. Deletion of TbGPI8, and inactivation of the GPI:protein transamidase complex, is tolerated by cultured procyclic forms. Using a conditional knockout, we show TbGPI8 is required for social motility (SoMo). This collective migration by cultured early procyclic forms has been linked to colonization of the tsetse fly digestive tract. The SoMo-negative phenotype was observed after a lag phase with respect to loss of TbGPI8 and correlated with an unexpectedly slow loss of procyclins, the major GPI-anchored proteins. Procyclins are not essential for SoMo, however, suggesting a requirement for at least one other GPI-anchored protein. Loss of TbGPI8 initiates the transition from early to late procyclic forms; this effect was observed in a subpopulation in suspension culture, and was more pronounced when cells were cultured on SoMo plates. Our results indicate two, potentially interlinked, scenarios that may explain the previously reported failure of TbGPI8 deletion mutants to establish a midgut infection in the tsetse fly: interference with stage-specific gene expression and absence of SoMo.

Elimination of GPI2 suppresses glycosylphosphatidylinositol GlcNAc transferase activity and alters GPI glycan modification in Trypanosoma brucei.

Many eukaryotic cell-surface proteins are post-translationally modified by a glycosylphosphatidylinositol (GPI) moiety that anchors them to the cell membrane. The biosynthesis of GPI anchors is initiated in the endoplasmic reticulum by transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol. This reaction is catalyzed by GPI GlcNAc transferase, a multisubunit complex comprising the catalytic subunit Gpi3/PIG-A as well as at least five other subunits, including the hydrophobic protein Gpi2, which is essential for the activity of the complex in yeast and mammals, but the function of which is not known. To investigate the role of Gpi2, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote and important model organism that initially provided the first insights into GPI structure and biosynthesis. We generated insect-stage (procyclic) trypanosomes that lack TbGPI2 and found that in TbGPI2-null parasites, (i) GPI GlcNAc transferase activity is reduced, but not lost, in contrast with yeast and human cells, (ii) the GPI GlcNAc transferase complex persists, but its architecture is affected, with loss of at least the TbGPI1 subunit, and (iii) the GPI anchors of procyclins, the major surface proteins, are underglycosylated when compared with their WT counterparts, indicating the importance of TbGPI2 for reactions that occur in the Golgi apparatus. Immunofluorescence microscopy localized TbGPI2 not only to the endoplasmic reticulum but also to the Golgi apparatus, suggesting that in addition to its expected function as a subunit of the GPI GlcNAc transferase complex, TbGPI2 may have an enigmatic noncanonical role in Golgi-localized GPI anchor modification in trypanosomes.

Complexity of the eukaryotic dolichol-linked oligosaccharide scramblase suggested by activity correlation profiling mass spectrometry.

The oligosaccharide required for asparagine (N)-linked glycosylation of proteins in the endoplasmic reticulum (ER) is donated by the glycolipid Glc3Man9GlcNAc2-PP-dolichol. Remarkably, whereas glycosylation occurs in the ER lumen, the initial steps of Glc3Man9GlcNAc2-PP-dolichol synthesis generate the lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) on the cytoplasmic side of the ER. Glycolipid assembly is completed only after M5-DLO is translocated to the luminal side. The membrane protein (M5-DLO scramblase) that mediates M5-DLO translocation across the ER membrane has not been identified, despite its importance for N-glycosylation. Building on our ability to recapitulate scramblase activity in proteoliposomes reconstituted with a crude mixture of ER membrane proteins, we developed a mass spectrometry-based 'activity correlation profiling' approach to identify scramblase candidates in the yeast Saccharomyces cerevisiae. Data curation prioritized six polytopic ER membrane proteins as scramblase candidates, but reconstitution-based assays and gene disruption in the protist Trypanosoma brucei revealed, unexpectedly, that none of these proteins is necessary for M5-DLO scramblase activity. Our results instead strongly suggest that M5-DLO scramblase activity is due to a protein, or protein complex, whose activity is regulated at the level of quaternary structure.

FTY720 and two novel butterfly derivatives exert a general anti-inflammatory potential by reducing immune cell adhesion to endothelial cells through activation of S1P(3) and phosphoinositide 3-kinase.

Sphingosine-1-phosphate (S1P) is a key lipid regulator of a variety of cellular responses including cell proliferation and survival, cell migration, and inflammatory reactions. Here, we investigated the effect of S1P receptor activation on immune cell adhesion to endothelial cells under inflammatory conditions. We show that S1P reduces both tumor necrosis factor (TNF)-α- and lipopolysaccharide (LPS)-stimulated adhesion of Jurkat and U937 cells to an endothelial monolayer. The reducing effect of S1P was reversed by the S1P1+3 antagonist VPC23019 but not by the S1P1 antagonist W146. Additionally, knockdown of S1P3, but not S1P1, by short hairpin RNA (shRNA) abolished the reducing effect of S1P, suggesting the involvement of S1P3. A suppression of immune cell adhesion was also seen with the immunomodulatory drug FTY720 and two novel butterfly derivatives ST-968 and ST-1071. On the molecular level, S1P and all FTY720 derivatives reduced the mRNA expression of LPS- and TNF-α-induced adhesion molecules including ICAM-1, VCAM-1, E-selectin, and CD44 which was reversed by the PI3K inhibitor LY294002, but not by the MEK inhibitor U0126.In summary, our data demonstrate a novel molecular mechanism by which S1P, FTY720, and two novel butterfly derivatives acted anti-inflammatory that is by suppressing gene transcription of various endothelial adhesion molecules and thereby preventing adhesion of immune cells to endothelial cells and subsequent extravasation.