The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development

Abstract

Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.

Keywords: E3 ubiquitin ligase; SCF-complex; Skp1; Toxoplasma gondii; cytoplasmic glycosylation; glycobiology; glycosyltransferase; parasitology.

FIGURE 1.

Orbitrap MS analysis of the TgSkp1 glycopeptide. RHΔΔ tachyzoites were lysed out of HFFs, urea-solubilized, and immunoprecipitated with bead-bound affinity-purified anti-TgSkp1 (pAb UOK75). The enriched preparation of TgSkp1 was eluted with triethylamine, reduced and alkylated, trypsinized, and analyzed by reverse phase-HPLC on an LTQ-XL Orbitrap MS. Extracted ion chromatograms showed coelution of a doubly charged (m/z 1436.6464) and a triply charged ion (m/z 958.0983) corresponding with a Δ mass of 0.56 ppm, to the predicted tryptic TgSkp1 peptide ¹⁴⁵IFNIVNDFT(HyP)EEEAQVR¹⁶¹ bearing a pentasaccharide with composition Hex3dHex1HexNAc1 (A). B, CID fragmentation of the doubly charged precursor ion yields a sequential loss of monosaccharide residues corresponding to Hex, Hex, dHex, Hex, and HexNAc, indicating the presence of a linear pentasaccharide. C, inspection of the full CID fragmentation spectrum shows b- (blue annotations) and y- (red annotations) ion series that match the predicted peptide sequence, as illustrated in the inset, and demonstrate that the glycan is linked via a hydroxylated derivative of Pro-154. Peptides with residual sugars are annotated in green.

FIGURE 2.

Comparative genomic and domain organization of Gnt1 and PgtA from Dictyostelium and Toxoplasma. A and B, exon-intron organization of the gnt1 and pgtA genes. gnt1 (A) and pgtA (B) gene models from Dictyostelium discoideum are from Refs. , and available at dictybase.org (27). The gene models from T. gondii (GT1 (type 1) strain) are from Ref. . The length from the start codon to the stop codon in nucleotides is in parentheses. C and D, domain organization of Gnt1 (C) and PgtA (D) proteins. Gnt1- and PgtA-like sequences from Toxoplasma and three other coccidian apicomplexans were aligned with corresponding sequences from D. discoideum and three other amoebozoa, and sequences from two chromerid alveolates, as shown in supplemental Figs. S1 and S2. Regions of high conservation among all 10 sequences are shown in color for the glycosyltransferase-like sequences and in gray for non-GT-like sequences. Toxoplasma sequences whose expression has been confirmed at the transcriptional (expressed sequence tags) or proteomic (MS) level are indicated in Ref. . Diagrams are shown to scale.

FIGURE 3.

Disruption and complementation of Tggnt1 and TgpgtA. A, strategy for deletion of Tggnt1 and its subsequent complementation. The plasmid-derived disruption of DNA with homologous targeting sequences was electroporated into parasites. Recovery of hxgprt-positive clones that were resistant to MPA and xanthine and were GFP-negative were candidates for double crossover gene replacement. B, gene replacement was confirmed by PCR-1, which demonstrated loss of gnt1 coding DNA, and PCR-2 and -3, which demonstrated that the inserted hxgprt DNA was flanked by neighboring gnt1 DNA. To complement Tggnt1 in the disruption strain, a plasmid containing an ∼7-kb genomic locus, including Tggnt1 coding region and 5′- and 3′-untranslated regions (A), was transfected. Complemented strains where the hxgprt is replaced by Tggnt1 locus were counter-selected under 6-thioxanthine. C, gnt1 replacement was confirmed by the positive PCR-1 and negative reactions for PCR-2 and PCR-3, which depended on the presence of hxgprt. D–F, TgpgtA was similarly targeted for disruption and complementation. Characteristics of the above strains are summarized in Table 1.

FIGURE 4.

Disruption of Tggnt1 or TgpgtA affects TgSkp1 glycosylation. Soluble S16 fractions from equivalent numbers (3 × 10⁶ cells) of parental RHΔΔ (RH) and RHphyAΔ-1 (phyA⁻), RHgnt1Δ, RHpgtAΔ, and their complemented cells were resolved by 4–12% SDS-PAGE, electroblotted, and probed using anti-TgSkp1 (UOK75) antiserum. Changes in glycosylation inferred from altered gel mobility were confirmed by mass spectrometry (Table 2). Similar results were obtained for independently derived clones of RHgnt1Δ and RHpgtAΔ.

FIGURE 5.

TgGnt1 is a Skp1 GlcNAcT. A, GlcNAcT activity in S100 cytosolic parasite was assayed based on transfer of ³H from 0.5 μm UDP-[³H]GlcNAc to exogenous HO-DdSkp1 for 1–3 h as described under “Experimental Procedures.” Reactions were loaded onto and separated on an SDS-polyacrylamide gel, and the Coomassie Blue-stained DdSkp1 bands were excised and subjected to liquid scintillation spectroscopy. The reaction time, presence of HO-Skp1, and source of the extract (RHΔΔ or RH, or RHgnt1Δ or gnt1Δ) were varied as indicated. B, entire lane from a parallel 3-h reaction (RH, +HO-Skp1) from A was analyzed for incorporation of ³H. Incorporation was only detected at the migration position of DdSkp1. C, donor substrate specificity of the GlcNAcT activity was examined by including a 9-fold excess of unlabeled UDP-GlcNAc or UDP-GalNAc to reactions containing 10 μm UDP-[³H]GlcNAc. Incorporation was measured as in A. Error bars show standard deviations of the mean of two replicates from each of two independent reactions. D, analysis of incorporated ³H. The reacted Skp1 band was excised from a PVDF membrane electroblot of the SDS-polyacrylamide gel, subjected to acid hydrolysis in 6 n HCl, and analyzed by high pH anion exchange chromatography. The hydrolysate was supplemented with GlcNH₂ and GalNH₂ and chromatographed on a Dionex PA-1 column. Elution of the sugar standards was monitored by a pulsed amperometric detector (nC), and fractions were collected to monitor the elution of ³H by scintillation counting (dpm).

FIGURE 6.

PgtA is a Skp1 GalT and FucT. A, GalT activity directed toward GlcNAc-Skp1 was assayed as described for GlcNAcT activity in Fig. 5A, except that GlcNAc-Skp1 and UDP-[³H]Gal were used in place of HO-Skp1 and UDP-[³H]GlcNAc. The reaction time, inclusion of GlcNAc-Skp1 and GDP-Fuc, and source of the extract (RHΔΔ or RH, or RHpgtAΔ or pgtAΔ) were varied as indicated. B, entire lane from a parallel 3-h reaction (RH, +Gn-Skp1) from A was analyzed for incorporation of ³H. Incorporation was only detected at the migration position of DdSkp1. C, [³H]DdSkp1 from the 3-h GalT reaction in A was isolated as in Fig. 5D and hydrolyzed in 4 m TFA. The hydrolysate was chromatographed on a Dionex PA-1 column with internal standards of Gal, Glc, Man, and Fuc, and the elution of ³H was monitored by scintillation counting of collected fractions. D–F, FucT activity assays. Reactions were conducted as above except that GDP-[³H]Fuc replaced UDP-[³H]Gal, and the dependence of incorporation on a 10-fold concentration excess of UDP-Gal, GlcNAc-Skp1, and PgtA in the extract and time was examined.

FIGURE 7.

Role of Tggnt1 and TgpgtA in parasite proliferation. HFF monolayers were inoculated with freshly isolated tachyzoite stage parasites at a multiplicity of infection of 0.002. After 5.5 days, monolayers were stained with crystal violet. A, representative images of cleared areas of the host monolayers. B–D, images digitized and plaque areas were calculated. The dot plots show the area distributions and average values ± S.E. from a representative of two independent experiments. Average parental strain areas ranged from 0.5 to 1.0 mm². p values for statistical significance of the differences, based on a one-way analysis of variance test, are shown above. ns = not significant. B, data from RHΔΔ, RHphyAΔ-1, and RHphyAΔ-2, generated by different strategies, RHgnt1Δ, and RHpgtAΔ strains. Bar graph shows average (±S.D.) from two independent experiments. C, data from strains in which Skp1 was SF-tagged. D, data from Tggnt1 or TgpgtA complemented (compl) strains.