Fucosebeta-1-P-Ser is a new type of glycosylation: using antibodies to identify a novel structure in Dictyostelium discoideum and study multiple types of fucosylation during growth and development

Abstract

Three antibodies that recognize distinct fucose epitopes were used to study fucosylation during growth and development of Dictyostelium discoideum. mAb83.5 is known to recognize an undefined "fucose epitope" on several proteins with serine-rich domains, while mAb CAB4, and a component of anti-horse-radish peroxidase, specifically recognize Fucalpha1,6GlcNAc and Fucalpha1,3GlcNAc residues respectively in the core of N-linked oligosaccharides. We show that mAb 83.5 defines a new type of O-glycosylation. Serine-containing peptides incubated with GDPbeta[3H]Fuc and microsomes formed two fucosylated products. A neutral product accounting for 30% of the label did not react with the antibody, while the rest of the label was incorporated into a charged product which contained all the mAb83.5 reactive material. beta-Elimination of the labeled peptide or endogenous products produced [3H]Fuc-1-P, indicating phosphodiester linkage to serine. Fucbeta-1-P and GDP-betaFuc at 100 microM blocked mAb83.5 binding to endogenous and peptide products, but their alpha-linked anomers did not. Electrospray ionization mass spectra of the neutral and anionic labeled products showed major peaks of mass units corresponding to O-Fuc-Ser peptide and O-Fuc-phospho-Ser peptide, respectively. The activity of Fuc-phosphotransferase exactly paralleled the accumulation of reactive glycans during growth and development. The expressions of N-glycan core Fucalpha1,6GlcNAc and Fucalpha1,3GlcNAc and their respective fucosyl transferase activities were also synchronous, but their developmental regulation differed from one another. Fucalpha1, 6GlcNAc was expressed maximally during growth but declined during development. In contrast core Fucalpha1,3GlcNAc epitopes were expressed almost exclusively during development. These findings provide direct evidence for a novel type of O-phosphofucosylation, demonstrate the existence of an O-fucosyl transferase, and identify two different types of core fucosylation in the N-glycans of Dictyostelium.