Toxoplasma gondii is a ubiquitous, obligate intracellular eukaryotic parasite that causes congenital birth defects, disease in immunocompromised individuals, and blindness. Protein glycosylation plays an important role in the infectivity and evasion of immune responses of many eukaryotic parasites and is also of great relevance to vaccine design. Here we demonstrate that micronemal protein 2 (MIC2), a motility-associated adhesin of T. gondii, has highly glycosylated thrombospondin repeat (TSR) domains. Using affinity-purified MIC2 and MS/MS analysis along with enzymatic digestion assays, we observed that at least seven C-linked and three O-linked glycosylation sites exist within MIC2, with >95% occupancy at these O-glycosylation sites. We found that addition of O-glycans to MIC2 is mediated by a protein O-fucosyltransferase 2 homolog (TgPOFUT2) encoded by the TGGT1_273550 gene. Even though POFUT2 homologs are important for stabilizing motility-associated adhesins and for host infection in other apicomplexan parasites, loss of TgPOFUT2 in T. gondii had only a modest impact on MIC2 levels and the wider parasite proteome. Consistent with this, both plaque formation and tachyzoite invasion were broadly similar in the presence or absence of TgPOFUT2. These findings indicate that TgPOFUT2 O-glycosylates MIC2 and that this glycan, in contrast to previous findings in another study, is dispensable in T. gondii tachyzoites and for T. gondii infectivity.
MS, glycosylation, fucosyltransferase, Proteomics, Toxoplasma gondii
NCBI PubMed ID: 30514763Publication DOI: 10.1074/jbc.RA118.005357Journal NLM ID: 2985121RPublisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Correspondence: tonkin@wehi.edu.au; goddard-borger.e@wehi.edu.au; nichollas.scott@unimelb.edu.au
Institutions: Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia, Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia, Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109, Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria 3010, Australia
Methods: SDS-PAGE, ELISA, Western blotting, serological methods, genetic methods, enzymatic digestion, invasion assays, LC-MS, LC-MS/MS, immunofluorescence analysis
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