Abstract

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.

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