A terminal α3-galactose modification regulates an E3 ubiquitin ligase subunit in Toxoplasma gondii

Abstract

<jats:p>Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is modified by a prolyl hydroxylase that mediates O<jats:sub>2</jats:sub> regulation of the social amoeba <jats:italic>Dictyostelium</jats:italic> and the parasite <jats:italic>Toxoplasma gondii</jats:italic>. The full effect of hydroxylation requires modification of the hydroxyproline by a pentasaccharide that, in <jats:italic>Dictyostelium</jats:italic>, influences Skp1 structure to favor assembly of Skp1/F-box protein subcomplexes. In <jats:italic>Toxoplasma</jats:italic>, the presence of a contrasting penultimate sugar assembled by a different glycosyltransferase enables testing of the conformational control model. To define the final sugar and its linkage, here we identified the glycosyltransferase that completes the glycan and found that it is closely related to glycogenin, an enzyme that may prime glycogen synthesis in yeast and animals. However, the <jats:italic>Toxoplasma</jats:italic> enzyme catalyzes formation of a Galα1,3Glcα linkage rather than the Glcα1,4Glcα linkage formed by glycogenin. Kinetic and crystallographic experiments showed that the glycosyltransferase Gat1 is specific for Skp1 in <jats:italic>Toxoplasma</jats:italic> and also in another protist, the crop pathogen <jats:italic>Pythium ultimum</jats:italic>. The fifth sugar is important for glycan function as indicated by the slow-growth phenotype of <jats:italic>gat1</jats:italic>Δ parasites. Computational analyses indicated that, despite the sequence difference, the <jats:italic>Toxoplasma</jats:italic> glycan still assumes an ordered conformation that controls Skp1 structure and revealed the importance of nonpolar packing interactions of the fifth sugar. The substitution of glycosyltransferases in <jats:italic>Toxoplasma</jats:italic> and <jats:italic>Pythium</jats:italic> by an unrelated bifunctional enzyme that assembles a distinct but structurally compatible glycan in <jats:italic>Dictyostelium</jats:italic> is a remarkable case of convergent evolution, which emphasizes the importance of the terminal α-galactose and establishes the phylogenetic breadth of Skp1 glycoregulation.</jats:p>

The nucleocytosolicO-fucosyltransferaseSpindlyaffects protein expression and virulence inToxoplasma gondii

“…1A) (32). As a positive control, UDP-Gal was hydrolyzed by a T. gondii galactosyltransferase (TgGat1), which adds a terminal galactose to the pentasaccharide attached to the T. gondii Skp1 E3 ubiquitin ligase subunit (33). Second, in the presence of GDP-Fuc, TgHis6SPY fucosylated itself (OFT activity in cis), as shown by Western blotting with AAL ( Fig.…”

“…We have not yet determined the effect of TgSPY knockout on cat infections, but we have observed that AAL-labeled assemblies are absent in the periphery of meiotic nuclei of sporulating oocysts (22). Finally, addition of O-Fuc is the second cytosolic glycosylation system present in T. gondii, which also adds a pentasaccharide to a hydroxyproline residue on Skp1, an E3 ubiquitin ligase subunit (33).…”

“…After washing the columns with 30 mM imidazole in 50 mM HEPES-NaOH (pH 7.4), 0.5 M NaCl, the SPY fraction was eluted with 250 mM imidazole in the same buffer, immediately dialyzed at 4° C against 50 mM HEPES-NaOH (pH 7.4), concentrated by centrifugal ultrafiltration, and frozen at -80 °C in aliquots. Samples were analyzed by SDS-PAGE and staining with Coomassie blue, or Western blotted and p probed with anti-His Ab, as described(33).GST-SRD expression in E. coliThe sequence encoding for the 79 aa at the N-terminus of TgGPN (TGGT1_285720) was used to generate a cDNA that was codon-optimized for E. coli and synthesized by GenScript to include BamHI and XhoI restriction sites. The SRD cDNA was cloned via BamHI/XhoI into pGEX6P-1, resulting in a N-terminalGST tag followed by the PreScission protease cleavage site and the SRD encoding sequence.…”

The nucleocytosolic O-fucosyltransferase SPINDLY affects protein expression and virulence in Toxoplasma gondii

“…1 A ) ( 32 ). As a positive control, UDP-Gal was hydrolyzed by a T. gondii galactosyltransferase ( Tg Gat1), which adds a terminal galactose to the pentasaccharide attached to the T. gondii Skp1 E3 ubiquitin ligase subunit ( 33 ). Second, in the presence of GDP-Fuc, His 6 Tg SPY fucosylated itself (OFT activity in cis ), as shown by western blotting with AAL ( Fig.…”

“…We have not yet determined the effect of Tg SPY knockout on cat infections, but we have observed that AAL-labeled assemblies are absent in the periphery of meiotic nuclei of sporulating oocysts ( 22 ). Finally, addition of O -Fuc is the second cytosolic glycosylation system present in T. gondii , which also adds a pentasaccharide to a hydroxyproline residue on Skp1, an E3 ubiquitin ligase subunit ( 33 ).…”