Glycosylation of human corticosteroid-binding globulin. Differential processing and significance of carbohydrate chains at individual sites.
Human corticosteroid-binding globulin (CBG) comprises 383 amino acids and six consensus sites for attachment of N-acetyllactosamine-type oligosaccharides. To study the extent of addition and processing of individual carbohydrate chains, we expressed CBG mutants, each containing only one of the six possible glycosylation sites, in Chinese hamster ovary cells and examined their electrophoretic, immunochemical, and lectin-binding properties. This indicated that Asn9, Asn308, and Asn347 are partially glycosylated and that oligosaccharides attached to Asn9, Asn238, Asn308, and Asn347 are predominantly biantennary, while more branched (most likely, triantennary) oligosaccharides are preferentially linked to Asn74 and Asn154. Only one of the biantennary chains (attached to Asn9) contains significant amounts of fucose. These data indicate that oligosaccharide processing is site-specific, and analyses of three other mutants, in which an additional glycosylation site was preserved, demonstrated that the processing of individual oligosaccharides occurs independently. Thus, the glycosylation of recombinant CBG appears to resemble that of natural human CBG. As we have previously found, glycosylation at Asn238 is essential for the production of CBG with steroid-binding activity, but when the mutant containing only one oligosaccharide at this position was enzymatically deglycosylated, its steroid-binding activity was unaltered. This suggests that interaction between this carbohydrate chain and the polypeptide is necessary for the folding and creation of the steroid-binding site only during CBG biosynthesis.
Avvakumov
G V
GV
MRC Group in Fetal and Neonatal Health and Development, University of Western Ontario, London, Canada.
Hammond
G L
GL
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D014156:Transcortin / Q000737:chemistry / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry
D018076:DNA, Complementary; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014364:Tryptophan; D014156:Transcortin