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Tag: Dictyostelium

Novel antibodies detect nucleocytoplasmic O-fucose in protist pathogens, cellular slime molds, and plants

Fig 1 Anti-fucopeptide antisera.

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.IMPORTANCEO-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.

Megna Tiwari, Elisabet Gas-Pascual, Manish Goyal, Marla Popov, Kenjiroo Matsumoto, Marianne Grafe, Ralph Gräf, Robert S Haltiwanger, Neil Olszewski, Ron Orlando, John C Samuelson, Christopher M West. mSphere. 2025 Feb 6:e0094524. doi: 10.1128/msphere.00945-24.

Synergy between a cytoplasmic vWFA/VIT protein and a WD40-repeat F-box protein controls development in Dictyostelium

Interactomes of FbxwD-FLAG3 and FLAG3Vwa1. Immunoprecipitations of FLAG tagged targets using anti-FLAG mAb M2 from cells solubilized in non-ionic detergent (0.2% NP-40) were subjected to a proteomics work-flow that included generation of peptides with endo Lys-C and trypsin followed by detection by nLC MS/MS and quantitation by spectral counting.

Like most eukaryotes, the pre-metazoan social amoeba Dictyostelium depends on the SCF (Skp1/cullin-1/F-box protein) family of E3 ubiquitin ligases to regulate its proteome. In Dictyostelium, starvation induces a transition from unicellular feeding to a multicellular slug that responds to external signals to culminate into a fruiting body containing terminally differentiated stalk and spore cells. These transitions are subject to regulation by F-box proteins and O2-dependent posttranslational modifications of Skp1. Here we examine in greater depth the essential role of FbxwD and Vwa1, an intracellular vault protein inter-alpha-trypsin (VIT) and von Willebrand factor-A (vWFA) domain containing protein that was found in the FbxwD interactome by co-immunoprecipitation. Reciprocal co-IPs using gene-tagged strains confirmed the interaction and similar changes in protein levels during multicellular development suggested co-functioning. FbxwD overexpression and proteasome inhibitors did not affect Vwa1 levels suggesting a non-substrate relationship. Forced FbxwD overexpression in slug tip cells where it is normally enriched interfered with terminal cell differentiation by a mechanism that depended on its F-box and RING domains, and on Vwa1 expression itself. Whereas vwa1-disruption alone did not affect development, overexpression of either of its three conserved domains arrested development but the effect depended on Vwa1 expression. Based on structure predictions, we propose that the Vwa1 domains exert their negative effect by artificially activating Vwa1 from an autoinhibited state, which in turn imbalances its synergistic function with FbxwD. Autoinhibition or homodimerization might be relevant to the poorly understood tumor suppressor role of the evolutionarily related VWA5A/BCSC-1 in humans.

Andrew W Boland, Elisabet Gas-Pascual, Hanke van der Wel, Hyun W Kim, Christopher M West. Front Cell Dev Biol. 2023 Sep 14;11:1259844. doi: 10.3389/fcell.2023.1259844. eCollection 2023.