Novel antibodies detect nucleocytoplasmic O-fucose in protist pathogens, cellular slime molds, and plants
Authors: Tiwari Megna , Gas-Pascual Elisabet , Goyal Manish , Popov Marla , Matsumoto Kenjiroo , Grafe Marianne , Graf Ralph , Haltiwanger Robert S. , Olszewski Neil , Orlando Ron , Samuelson John , West Christopher M. 
Published: October 2024

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 pathogenToxoplasma gondii, the social amoebaDictyostelium, and higher plants, where it is called Spy because mutants have a spindly appearance. O-fucosylation, which is required for optimal proliferation ofToxoplasmaandDictyostelium, 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 inToxoplasmausingAleuria aurantialectin, 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 inToxoplasma, Dictyostelium, andArabidopsis, as well as O-Fuc occurring on secretory proteins ofDictyosteliumand mammalian cells, although the latter are frequently blocked by further glycosylation. The antibodies labelToxoplasma,Acanthamoeba, andDictyosteliumin a pattern reminiscent of O-GlcNAc in animal cells including nuclear pores. The O-fucome ofDictyosteliumis partially conserved with that ofToxoplasmaand 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.

IMPORTANCE

O-fucose, a form of mono-glycosylation on serine and threonine residues of nuclear and cytoplasmic proteins of some parasites, other unicellular eukaryotes, and plants, is understudied because it is difficult to detect owing to its neutral charge and lability during mass spectrometry. Yet the O-fucosyltransferase enzyme (OFT) is required for optimal growth of the agent for toxoplasmosis,Toxoplasma gondii, and an unrelated protist, the social amoebaDictyostelium discoideum. Furthermore, O-fucosylation is closely related to the analogous process of O-GlcNAcylation of thousands of proteins of animal cells, where it plays a central role in stress and nutritional responses. O-Fuc is currently best detected usingAleuria aurantialectin (AAL), but in most organisms AAL also recognizes a multitude of proteins in the secretory pathway that are modified with fucose in different ways. By establishing the potential to induce highly specific rabbit antisera that discriminate O-Fuc from all other forms of protein fucosylation, this study expands knowledge about the protist O-fucome and opens a gateway to explore the potential occurrence and roles of this intriguing posttranslational modification in bacteria and other protist pathogens such asAcanthamoeba castellanii.