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 , Gräf Ralph , Haltiwanger Robert S. , Olszewski Neil , Orlando Ron , Samuelson John C. , West Christopher M. 
Published: February 2025

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-fucose). A nucleocytoplasmic O-fucosyltransferase 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 are involved in stress and nutritional responses. O-fucose 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-fucose occurring on secretory proteins of
Dictyostelium
and mammalian cells except when 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-fucose, document the conservation of the O-fucome among unrelated protists, and enable the study of the O-fucomes of other organisms possessing O-fucosyltransferase-like genes.

IMPORTANCE

O-fucose (O-Fuc), 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 amoeba
Dictyostelium 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 using
Aleuria aurantia
lectin (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 as
Acanthamoeba castellanii
.