Glycosylation of human corticosteroid-binding globulin at aspargine 238 is necessary for steroid binding.
Human corticosteroid binding-globulin (CBG) is a plasma glycoprotein that binds and regulates the biological activity of glucocorticoids and progesterone. Carbohydrates comprise approximately 25% of its molecular mass being represented by bi- and triantennary N-linked oligosaccharides of the N-acetyllactosamine type. To assess the impact of these carbohydrate chains on CBG production and steroid binding, we mutated a human CBG cDNA so that the six consensus sites for N-glycosylation in the CBG polypeptide were eliminated individually and in various combinations. Expression of the mutant cDNAs in Chinese hamster ovary cells showed that all consensus sites may be utilized during the CBG biosynthesis and that the immunochemical properties of the recombinant glycoproteins are similar to those of CBG isolated from human serum. Removal of sugar chains generally led to a reduction in the secretion of recombinant CBG, but complete removal of N-glycosylation sites did not prevent production or secretion of the protein. Our data indicate that an oligosaccharide linked to Asn238 is essential for steroid binding, and we suggest that an interaction between this sugar chain and the polypeptide may be essential for the creation of a high affinity steroid-binding site. In addition, concanavalin A chromatography of mutants containing only one N-glycosylation site at either Asn74 or Asn238 indicated that processing of the oligosaccharides at these positions is site-specific.
Avvakumov
G V
GV
Medical Research Council Group in Fetal and Neonatal Health and Development, University of Western Ontario, London, Canada.
Warmels-Rodenhiser
S
S
Hammond
G L
GL
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D006854:Hydrocortisone / Q000378:metabolism; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014156:Transcortin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D011994:Recombinant Proteins; D001216:Asparagine; D014156:Transcortin; D006854:Hydrocortisone