Glycobiology, Vol 8, 799-811, Copyright © 1998 by Society for Glycobiology
G Srikrishna, L Wang and HH Freeze
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.
ORIGINAL ARTICLES
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
The Burnham Institute, La Jolla Cancer Research Center, La Jolla, California 92037, USA.
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