Expression of biologically active human corticosteroid binding globulin by insect cells: acquisition of function requires glycosylation and transport.
Human corticosteroid binding globulin (hCBG) is a 50- to 55-kDa serum glycoprotein that binds cortisol and progesterone with high affinity. To map the steroid-binding domain and to investigate the folding pathways of hCBG, we have established an expression system based on infection of insect cells with a recombinant baculovirus encoding hCBG. Infected Spodoptera frugiperda (Sf9) cells secrete immunoreactive hCBG at high levels (16-24 pmol per 10(6) cells per 40 h), and the recombinant protein binds cortisol with an affinity and specificity equivalent to that of human serum-derived hCBG. Thus, this system has the potential to provide large amounts of wild-type and mutant hCBGs for physical-chemical analysis. Cotranslational asparagine-linked glycosylation is essential for acquisition of steroid-binding capability, as shown by the lack of cortisol-binding activity of unglycosylated hCBG secreted in the presence of tunicamycin. Golgi-associated oligosaccharide processing, however, is not required for activity, as demonstrated by the endoglycosidase H susceptibility of the fully active, secreted glycoprotein. Comparison of the steroid-binding properties of intracellular and secreted hCBG with that synthesized in vitro in the rabbit reticulocyte lysate system suggests that this protein undergoes a maturation process during transport through the secretory pathway. This system will be useful for identifying the molecular determinants of biological function in hCBG.
Ghose-Dastidar
J
J
Department of Biochemistry, Mount Sinai School of Medicine, New York, NY 10029.
Ross
J B
JB
Green
R
R
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D006854:Hydrocortisone / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013256:Steroids / Q000378:metabolism; D014156:Transcortin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D013256:Steroids; D014156:Transcortin; D006854:Hydrocortisone