Browsing by Author "Choi, Jung Min"
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Item Functional characterization of Mucin-Associated Surface Protein (MASP) in the human parasite Trypanosoma cruzi(2012) Choi, Jung Min; El-Sayed, Najib M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)MASPs are members of a multigenic family recently identified during the sequencing of the T. cruzi CL Brener genome. This family contains around 1,400 members, consisting of approximately 6% of the whole coding genes. Highly conserved N- and C-terminal domains, which encode a signal peptide and GPI-anchor addition site respectively, and a hypervariable central region, characterize MASPs. Members of this family are predominantly expressed in the infective trypomastigote form. We hypothesized that members of the T. cruzi MASP protein family play a major role in the interaction of the parasite with the host cell. In order to investigate a putative role for T. cruzi MASP at the host-pathogen interface, we have used MASP as a bait protein against the human proteome using a high-throughput platform that we have recently established for identifying protein-protein interactions between pathogens and theirs hosts. Yeast two-hybrid screens identified human SNAPIN as one of two major MASP interacting proteins. SNAPIN is a member of the SNARE protein complex, which may have a role in a calcium-dependent exocytosis. The MASP-SNAPIN interaction was further validated using in vivo co-Affinity Purification and in vitro pull-down assays. Immunofluorescence assays showed human SNAPIN is recruited to the parasite surface during invasion. Co-localization experiments indicated that SNAPIN is associated with the late endosomes and lysosomes. Supporting our initial hypothesis, SNAPIN depletion using siRNA oligomers in HeLa cells and snapin-/- in Mouse Embryonic Fibroblast (MEF) cells significantly inhibited T. cruzi invasion, suggesting a role for SNAPIN in this process. Lysosomes in snapin-/- MEF cells displayed aberrant morphology and distribution and the parasites did not recruit host lysosomes efficiently when compared to wild-type cells. This was likely due to an impaired calcium-dependent lysosome exocytosis in snapin-/- MEF cells. SNAPIN was translocated to the plasma membrane upon calcium influx induced by a calcium ionophore (Ionomycin), resulting in the exposure of the luminal domain of SNAPIN to the extracelluar space. Leishmania tarentolae transgenic strains expressing two different MASP proteins were shown to trigger intracellular calcium transients in HeLa cells, presumably by injuring the cell membrane. We propose that T. cruzi MASP plays a role in wounding the plasma membrane of the host cell, which in turn elicits a transient intracellular calcium flux and leads to the translocation of lysosome-associated SNAPIN to the plasma membrane. Human SNAPIN, through its exposed luminal domain would then provide an anchor for the entry to the parasite into the cell. The mechanism of T. cruzi MASP evoked calcium influx in the host cell membrane remains under investigation.