POFUT2

POFUT2 is the protein O-fucosyltransferase of Toxoplasma gondii (encoded by TGGT1_273550) that installs O-linked fucose on the thrombospondin type I repeat (TSR) domains of the microneme adhesin MIC2 within the secretory pathway (PMID:30538131, PMID:30514763). Genetic disruption abolishes MIC2 O-fucosylation, and mass spectrometry shows that at least three TSR acceptor sites are modified with high (>95%) occupancy by a GlcFuc (dHexHex) disaccharide, a modification that co-occurs with C-mannosylation of TSR tryptophans on the same protein (PMID:30514763, PMID:30538131). This O-fucosylation depends on GDP-fucose delivery into the secretory pathway by the transporter NST2, whose disruption phenocopies loss of POFUT2 (PMID:30538131). Functionally, POFUT2-mediated O-fucosylation supports MIC2 stability and trafficking to the micronemes: loss of the enzyme reduces cellular MIC2 and causes a fraction of MIC2 to accumulate earlier in the secretory pathway (PMID:30538131). The consequence for host cell invasion is divergent across studies, with one reporting an invasion and attachment defect comparable to Δmic2 (PMID:30538131) and another finding little impact on invasion or plaque formation (PMID:30514763).

1. 2018 High

   Establishing that POFUT2 is the enzyme responsible for MIC2 TSR O-fucosylation answered what glycosyltransferase generates this modification and linked it to MIC2 processing and trafficking.

   Evidence Δpofut2 knockout with MS glycan analysis, anti-GlcFuc antibody detection, and immunolocalization in T. gondii

   PMID:30538131

   Open questions at the time

   • Structural basis of substrate recognition not resolved
   • Mechanism by which O-fucose promotes MIC2 folding/trafficking not defined
2. 2018 High

   Mapping the glycan structures defined the chemistry of the modification, showing GlcFuc disaccharide addition at TSR acceptor sites co-occurring with TSR Trp C-mannosylation.

   Evidence MS/MS glycan site mapping and enzymatic digestion of affinity-purified MIC2 across two independent labs

   PMID:30538131 PMID:30514763

   Open questions at the time

   • Whether C-mannosylation and O-fucosylation are functionally interdependent not tested
   • Full inventory of POFUT2 substrates beyond MIC2 not determined
3. 2018 Medium

   Disruption of NST2 placed POFUT2 in a GDP-fucose-dependent pathway, identifying the upstream nucleotide-sugar supply required for the modification.

   Evidence Δnst2 knockout with antibody-based O-fucosylation detection and invasion assay in T. gondii

   PMID:30538131

   Open questions at the time

   • Direct demonstration of NST2 GDP-fucose transport activity not shown
   • Whether NST2 serves other fucosyltransferases not addressed
4. 2018 Medium

   Phenotyping Δpofut2 addressed whether O-fucosylation is required for host cell invasion, but the two studies reached divergent conclusions on the severity of the invasion defect.

   Evidence Independent Δpofut2 knockouts with attachment, invasion, plaque and dissemination assays in human foreskin fibroblasts

   PMID:30538131 PMID:30514763

   Open questions at the time

   • Source of the invasion-phenotype discrepancy between labs unresolved
   • Contribution of residual MIC2 versus trafficking defect to invasion not separated

• The molecular mechanism by which O-fucosylation stabilizes and traffics MIC2, and the reason for the discordant invasion phenotypes, remain unresolved.
• No structural model of POFUT2-TSR recognition
• Full substrate repertoire beyond MIC2 unknown
• Basis of cross-study invasion discrepancy undetermined