Proteomics and Glycomics Analyses of N-Glycosylated Structures Involved in Toxoplasma gondii-Host Cell Interactions

Abstract

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man 5-8 -(GlcNAc) 2 ) and paucimannosidic (Man 3-4 (GlcNAc) 2 ) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii. Molecular & Cellular Proteomics 7:891-910, 2008.

Suggestive Evidence for Darwinian Selection against Asparagine-Linked Glycans of Plasmodium falciparum and Toxoplasma gondii

“…5D). In contrast, the plant lectin concanavalin A, which has previously been used to visualize Toxoplasma glycoproteins with N-glycans (16,32), binds to any mannose-containing glycan (Fig. 5E).…”

“…Because protein prediction is difficult for Toxoplasma and Eimeria, which contain many introns in their genes, TBASTN was also used to search the contigs and/or expressed sequence tags (ESTs) of these organisms (17,49). Figure 1 was drawn based upon the predicted sets of N-glycans made by these apicomplexan parasites, rather than those that are experimentally determined (which might include host N-glycan precursors in the case of Toxoplasma) (16,19).…”

“…(ii) There is also controversy concerning whether the Nglycans of Toxoplasma, after removal of Glc by glucosidases in the ER lumen, contain either 7 sugars (Man 5 GlcNAc 2 ), like Entamoeba (32,33), or 11 sugars (Man 9 GlcNAc 2 ), like the human host (16,19,26). If it is Man 5 GlcNAc 2 , then Toxoplasma uses the dolichol-PP-linked glycan predicted by its set of Alg enzymes (32,46).…”

“…If it is Man 5 GlcNAc 2 , then Toxoplasma uses the dolichol-PP-linked glycan predicted by its set of Alg enzymes (32,46). If it is Man 9 GlcNAc 2 , then Toxoplasma uses the dolichol-PP-linked glycan of the host cell (16,19,26).…”

Comprehensive Proteome Analysis of the Excretory/Secretory Proteins of Toxoplasma gondii

“…Many proteins were present in multiple spots, consistent with the presence of post-translational modifications. 25,26 In a similar study, A23187 used to stimulate calcium mediated ESP from T. gondii. As a result, total of 213 protein spots were identified by 2DE.…”