The role of glycosylation in the biosynthesis and acquisition of ligand-
binding activity of the folate-binding protein in cultured KB cells
CA Luhrs
Department of Medicine, State University of New York-Health Science Center.
The biosynthesis, processing, and ligand-binding function of the
membrane-associated and soluble forms of the folate-binding protein (FBP)
in KB cells, a cultured human cell line, were studied using pulse- chase
labeling with [35S] methionine. The intermediary and mature forms of the
protein were isolated by immunoprecipitation and affinity chromatography
and analyzed by sodium dodecyl sulfate electrophoresis and autoradiography.
The earliest species identified had an Mr of 32 Kd and disappeared over 5
hours concomitant with the appearance of a 38-Kd cellular FBP. As the 38-Kd
species disappeared, a 40-Kd form appeared in the medium. When tunicamycin
was added to the culture medium to inhibit core glycosylation, a 26-Kd
aglycosylated species and minor 28- Kd and 30-Kd forms appeared.
Endoglycosidase H, which cleaves high mannose but not complex
oligosaccharides, reduced the 32-Kd species to 26-Kd but the enzyme had no
effect on the 38-Kd form, indicating that this species is complex
glycosylated. Monensin, which blocks complex glycosylation, also inhibited
synthesis of the 38-Kd species. Although both the 32-Kd and 38-Kd forms had
ligand-binding sites (as demonstrated by binding to a folate-Sepharose
matrix), the 26-Kd aglycosylated species, labeled in the presence of
tunicamycin, lacked similar binding sites because it did not bind to the
affinity matrix. In contrast, the aglycosylated 26-Kd form, which was
obtained by treatment of the 32-Kd species with endoglycosidase H, did bind
to the folate affinity matrix, indicating that it retained ligand-binding
function. Thus, the high mannose oligosaccharide moiety is not required for
the folate-binding property of the FBP, but its addition to the polypeptide
chain precedes a later step that is necessary for the mature protein to
have ligand-binding function.
Volume 77,
Issue 6,
pp. 1171-1180,
03/15/1991
Copyright © 1991 by The American Society of Hematology
