A glycogenin homolog controls Toxoplasma gondii growth via glycosylation of an E3 ubiquitin ligase

Skp1, a subunit of E3-Skp1/Cullin-1/F-box protein ubiquitin ligases, is uniquely modified in protists by an O2-dependent prolyl hydroxylase, which forms the attachment site for a novel pentasaccharide. Mutational studies demonstrate the importance of the core glycan for growth of the parasite Toxoplasma gondii in fibroblasts, but the significance of the non-reducing terminal sugar is unknown. Here we investigated a homolog of glycogenin, an enzyme that can initiate and prime glycogen synthesis in yeast and animals. Gat1 is required for pentasaccharide assembly in cells and catalyzes the addition of an α-galactose in 3-linkage to the subterminal α3-linked glucose residue in vitro. A strong selectivity of Gat1 for Skp1 in extracts is consistent with evidence that Skp1 is the sole target of the glycosyltransferase pathway. gat1-disruption resulted in slow growth indicating the importance of the complete glycan. Molecular dynamics simulations suggested that the full glycan helps organize Skp1 as previously described in the amoebozoan Dictyostelium where a distinct glycosyltransferase assembles a different terminal disaccharide. The crystal structure of Gat1 from the plant pathogen Pythium ultimum confirmed the striking similarity to glycogenin, with differences in the active sites providing an explanation for its distinct substrate preference and regiospecificity. Gat1 also exhibited low α-glucosyltransferase activity like glycogenin, but autoglycosylation was not detected and gat1-disruption revealed no effect on starch accumulation in Toxoplasma. A phylogenetic analysis suggested that Gat1 was a progenitor of glycogenin, and acquired its role in glycogen formation following the ancestral disappearance of the underlying Skp1 glycosyltransferase Glt1 prior to amoebozoan evolution.