Abstract
Toxoplasma gondii motility, which is essential for host cell entry, migration through host tissues, and invasion, is a unique form of actin-dependent gliding. It is powered by a motor complex mainly composed of myosin heavy chain A, myosin light chain 1, gliding associated proteins GAP45, and GAP50, the only integral membrane anchor so far described. In the present study, we have combined glycomic and proteomic approaches to demonstrate that all three potential N-glycosylated sites of GAP50 are occupied by unusual N-glycan structures that are rarely found on mature mammalian glycoproteins. Using site-directed mutagenesis, we show that N-glycosylation is a prerequisite for GAP50 transport from the endoplasmic reticulum to the Golgi apparatus and for its subsequent delivery into the inner complex membrane. Assembly of key partners into the gliding complex, and parasite motility are severely impaired in the unglycosylated GAP50 mutants. Furthermore, comparative affinity purification using N-glycosylated and unglycosylated GAP50 as bait identified three novel hypothetical proteins including the recently described gliding associated protein GAP40, and we demonstrate that N-glycans are required for efficient binding to gliding partners. Collectively, these results provide the first detailed analyses of T. gondii N-glycosylation functions that are vital for parasite motility and host cell entry.
Highlights
From the ‡Center for Infection and Immunity of Lille, CNRS UMR 8204, INSERM U 1019, Institut Pasteur de Lille, Universite Lille Nord de France, 59000 Lille, France; §Laboratoire de Spectrometrie de Masse Bioorganique, IPHC, CNRS UMR7178, 67087 Strasbourg, France; ¶Unitede Glycobiologie Structurale et Fonctionnelle, CNRS UMR 8576, IFR 147, Universitedes Sciences et Technologies de Lille 1, 59655 Villeneuve d’Ascq, France; ʈLaboratoire de Physiologie Cellulaire, INSERM U 1003, Universitedes Sciences et Technologies de Lille 1, 59655 Villeneuve d’Ascq, France
We chose the heterologous GRA1 promoter because the weak expression driven by GAP50 promoter yields an insufficient amount of protein for further classical glycomic and proteomic analyses
This confirmed that the ectopically expressed GAP50-HAFAG protein was strongly detected in the inner membrane complex, which was stained with monoclonal antibodies specific to IMC1, a major component of the subpellicular network that tightly apposes the cytoplasmic face of the IMC [29]
Summary
Culture of Parasites—Human foreskin fibroblasts (HFFs) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal calf serum (FCS, Invitrogen), 2 mM L-glutamine, Invitrogen) and 10 000 U/ml Penicillin-Streptomycin (Invitrogen). Comparative proteomic analyses were performed using lysates from equal numbers (1010) of parasites from stable wild type expressing triple N-glycosylated GAP50-HAFLAG tachyzoites and completely N-deglycosylated GAP50-HAFLAG parasites In this case, the affinity chromatography was performed under higher salt stringency using TBS containing 0.5 M NaCl and bound proteins were eluted with 0.1 M glycine.HCl pH 2.5 and neutralized with 0.1 M Tris.HCl pH 9.4. Trails of gliding parasites were visualized with a Zeiss Axiophot microscope These treated or untreated wild type, ectopic expresser and completely deglycosylated GAP50 expressing parasites were resuspended with DMEM medium containing 10% FCS, 2 mM L-glutamine and 10 000 U/ml Penicillin-Streptomycin, loaded onto confluent monolayer HFF cells and grown for 24 h at 37 °C. The intracellular parasites were fixed and stained with monoclonal antibody specific to SAG1 for immunofluorescence as above and intracellular tachyzoites were counted under Zeiss Axiophot microscopy
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