Abstract
Cellular adaptations to change often involve post-translational modifications of nuclear and cytoplasmic proteins. An example found in protists and plants is the modification of serine and threonine residues of dozens to hundreds of nucleocytoplasmic proteins with a single fucose (O-Fuc). A nucleocytoplasmic O-fucosyltransferase (OFT) occurs in the pathogen Toxoplasma gondii, the social amoeba Dictyostelium, and higher plants, where it is called Spy because mutants have a spindly appearance. O-fucosylation, which is required for optimal proliferation of Toxoplasma and Dictyostelium, is paralogous to the O-GlcNAcylation of nucleocytoplasmic proteins of plants and animals that is involved in stress and nutritional responses. O-Fuc was first discovered in Toxoplasma using Aleuria aurantia lectin, but its broad specificity for terminal fucose residues on N- and O-linked glycans in the secretory pathway limits its use. Here we present affinity purified rabbit antisera that are selective for the detection and enrichment of proteins bearing fucose-O-Ser or fucose-O-Thr. These antibodies detect numerous nucleocytoplasmic proteins in Toxoplasma, Dictyostelium, and Arabidopsis, as well as O-Fuc occurring on secretory proteins of Dictyostelium and mammalian cells, although the latter are frequently blocked by further glycosylation. The antibodies label Toxoplasma, Acanthamoeba, and Dictyostelium in a pattern reminiscent of O-GlcNAc in animal cells including nuclear pores. The O-fucome of Dictyostelium is partially conserved with that of Toxoplasma and is highly induced during starvation-induced development. These antisera demonstrate the unique antigenicity of O-Fuc, document conservation of the O-fucome among unrelated protists, and will enable the study of the O-fucomes of other organisms possessing OFT-like genes.