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, 294 (5), 1541-1553

Protein O-fucosyltransferase 2-mediated O-glycosylation of the Adhesin MIC2 Is Dispensable for Toxoplasma gondii Tachyzoite Infection

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Protein O-fucosyltransferase 2-mediated O-glycosylation of the Adhesin MIC2 Is Dispensable for Toxoplasma gondii Tachyzoite Infection

Sachin Khurana et al. J Biol Chem.

Abstract

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.

Keywords: MS; Toxoplasma gondii; fucosyltransferase; glycosylation; proteomics.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Establishment and validation of the M2AP SF-TAP T. gondii line. A, addition of a C-terminally appended SF-TAP tag enables detection of M2AP within the T. gondii line M2AP SF-TAP compared with the parental line. B, M2AP, detected using α-FLAG, co-localizes with MIC2 within intracellular tachyzoites. Scale bar corresponds to 5 μm. C, enrichment of M2AP SF-TAP tagged protein enables the isolation of the MIC2-M2AP complex to high purity, as determined by Coomassie-stained gels (image reproduced from Fig. 4C).
Figure 2.
Figure 2.
Characterization of MIC2 glycosylation events. A, within MIC2, ten glycosylation sites were identified, corresponding to seven C-glycosylation events and three GlcFuc sites of modification. The residues modified and the corresponding carbohydrate modification are mapped to the MIC2 sequence. All observed glycosylation events lie within the TSR domains. B, peptides containing observed the C-glycosylation events Trp276, Trp279, Trp348, Trp351, Trp479, Trp537 and Trp540.
Figure 3.
Figure 3.
Sites of O-fucosylations of MIC2 are modified at a high occupancy. A, analysis of tryptic digested MIC2 with EThcD fragmentation enabled identification of the C-glycosylated and GlcFuc within 266TLPQDAICSDWSAWSPCSVSCGDGSQIR293, with the site of GlcFuc localized to S285. B, analysis of trypsin- and GluC-digested MIC2 with EThcD fragmentation enabled identification of glycosylated 479WSTCSVSCGGGLK491, with the site of GlcFuc localized to Ser485. Analysis of GluC-digested MIC2 using a combination of HCD (C) and EThcD (D) fragmentation enabled localization of GlcFuc to Thr546 on the glycopeptide 543CSVTCGDGVRE553. The GlcFuc-containing glycoforms of these peptides were the most abundant forms observed (E–G), supporting the hypothesis that these sites are occupied at high occupancy.
Figure 4.
Figure 4.
TGGT1_273550 encodes T. gondii POFUT2. A, endogenously tagging TgPOFUT2 with HA enabled detection in tachyzoites. This band is absent in a Δtgpofut2 line. B, loss of POFUT2 leads to a change in the migration of MIC2 and a small, ∼20% decrease in MIC2. Data points refer to individual biological replicates, showing mean ± S.E. p values were calculated using a paired t test; **, p <0.01. C, Coomassie-stained gel of isolation of M2AP enables co-isolation of MIC2 in the Δtgpofut2 line at comparable levels, with B1 and B2 corresponding to two independent isolations from biological replicates. D, for the peptide 266TLPQDAICSDWSAWSPCSVSCGDGSQIR293, unmodified, single, and doubled C-glycosylated peptides from MIC2 were identified, but no GlcFuc-containing species were observed. Extracted ion chromatograms demonstrate that these identified forms are clearly detectible in panels 1, 5, and 6, whereas even at the MS1 level, no ions corresponding to the GlcFuc glycoforms were observed in panels 2–4.
Figure 5.
Figure 5.
Quantitative proteomic analysis of Δtgpofut2 compared with the parental line. Label-free quantification of isolated tachyzoites was undertaken to compare Δtgpofut2 with the parental line. A, identified proteins are presented as a volcano plot depicting mean LFQ intensity ratios of Δtgpofut2 versus the parental line plotted against logarithmic t test p values from five biological experiments of each line. B, heat map of z-scored values of the proteins observed to change between the Δtgpofut2 and the parental line, demonstrating the consistency of these changes across experiments.
Figure 6.
Figure 6.
Phenotypic analysis of Δtgpofut2 compared with the parental line. A, i, morphological assessment of plaque assays comparing the parental line (TgM2AP-SF TAP), TgPOFUT2-HA, Δtgpofut2 and Δmic2). ii, numerical assessment of plaque size compared with the TgM2AP-SF TAP parental line and Δmic2. iii, numerical assessment of plaque capacity, as a surrogate of host cell attachment, compared across all lines. B, IFA assessment of localization of MIC2, detected using αMIC2, and M2AP, detected using αFLAG, suggests that O-glycosylation plays no detectable role in protein trafficking. Scale bars correspond to 5 μm. C, invasion assays at 2, 10, and 30 min demonstrate no detectable difference in invasion capacity. Each data point represents the average value across a biological replicate and are collectively represented using mean ± S.E. p values were calculated using a one-way analysis of variance for A, ii and iii, and two-way analysis of variance for C; **, p <0.01; ****, p <0.0001.

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