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Toxoplasma gondii is an intracellular parasite that causes disseminated infections that can produce neurological damage in fetuses and immunocompromised individuals. Microneme protein 2 (MIC2), a member of the thrombospondin-related anonymous protein (TRAP) family, is a secreted protein important for T. gondii motility, host cell attachment, invasion, and egress. MIC2 contains six thrombospondin type I repeats (TSRs) that are modified by C -mannose and O -fucose in Plasmodium spp. and mammals. Here, using MS analysis, we found that the four TSRs in T. gondii MIC2 with protein O -fucosyltransferase 2 (POFUT2) acceptor sites are modified by a dHexHex disaccharide, whereas Trp residues within three TSRs are also modified with C -mannose. Disruption of genes encoding either POFUT2 or the putative GDP-fucose transporter (NST2) resulted in loss of MIC2 O -fucosylation, as detected by an antibody against the GlcFuc disaccharide, and in markedly reduced cellular levels of MIC2. Furthermore, in 10-15% of the Δ pofut2 or Δ nst2 vacuoles, MIC2 accumulated earlier in the secretory pathway rather than localizing to micronemes. Dissemination of tachyzoites in human foreskin fibroblasts was reduced for these knockouts, which both exhibited defects in attachment to and invasion of host cells comparable with the Δ mic2 phenotype. These results, indicating that O -fucosylation of TSRs is required for efficient processing of MIC2 and for normal parasite invasion, are consistent with the recent demonstration that Plasmodium falciparum Δ pofut2 strain has decreased virulence and also support a conserved role for this glycosylation pathway in quality control of TSR-containing proteins in eukaryotes., (© 2019 Bandini et al.)

Toxoplasma gondii parasites must actively invade host cells to propagate. Secretory microneme proteins have been shown to be important for both gliding motility and active invasion. MIC2-M2AP is a protein complex that is essential for productive motility and rapid invasion by binding to host cell surface receptors. To investigate the architecture of the MIC2 and M2AP complex, we identified the minimal domains sufficient for interaction and solved the NMR solution structure of the globular domain of M2AP. We found that M2AP adopts a modified galectin fold similar to the C-terminal domain of another microneme protein, MIC1. NMR and immunoprecipitation analyses implicated hydrophobic residues on one face of the M2AP galectin fold in binding to the membrane proximal sixth thrombospondin type I repeat domain of MIC2. Our findings provide a second example of a galectin fold adapted for microneme protein-protein interactions and suggest a conserved strategy for the assembly and folding of diverse protein complexes., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)