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

Three antibodies that recognize distinct fucose epitopes were used to studyfucosylation during growth and development of Dictyostelium discoideum.mAb83.5 is known to recognize an undefined "fucose epitope" on severalproteins with serine-rich domains, while mAb CAB4, and a component ofanti-horse-radish peroxidase, specifically recognize Fucalpha1,6GlcNAc andFucalpha1,3GlcNAc residues respectively in the core of N-linkedoligosaccharides. We show that mAb 83.5 defines a new type ofO-glycosylation. Serine-containing peptides incubated with GDPbeta[3H]Fucand microsomes formed two fucosylated products. A neutral productaccounting for 30% of the label did not react with the antibody, while therest of the label was incorporated into a charged product which containedall the mAb83.5 reactive material. beta- Elimination of the labeled peptideor endogenous products produced [3H]Fuc-1-P, indicating phosphodiesterlinkage to serine. Fucbeta-1-P and GDP-betaFuc at 100 microM blockedmAb83.5 binding to endogenous and peptide products, but their alpha-linkedanomers did not. Electrospray ionization mass spectra of the neutral andanionic labeled products showed major peaks of mass units corresponding toO-Fuc-Ser peptide and O-Fuc-phospho-Ser peptide, respectively. The activityof Fuc- phosphotransferase exactly paralleled the accumulation of reactiveglycans during growth and development. The expressions of N-glycan coreFucalpha1,6GlcNAc and Fucalpha1,3GlcNAc and their respective fucosyltransferase activities were also synchronous, but their developmentalregulation differed from one another. Fucalpha1, 6GlcNAc was expressedmaximally during growth but declined during development. In contrast coreFucalpha1,3GlcNAc epitopes were expressed almost exclusively duringdevelopment. These findings provide direct evidence for a novel type ofO-phosphofucosylation, demonstrate the existence of an O- fucosyltransferase, and identify two different types of core fucosylation in theN-glycans of Dictyostelium.