Document Type

Article

Publication Title

Proceedings of the National Academy of Sciences of the United States of America

Publisher

National Academy of Sciences

Publication Date

8-1-1991

Volume

88

Issue

15

Disciplines

Biochemistry | Chemistry | Life Sciences | Physical Sciences and Mathematics

Abstract

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 106 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.

Rights

© 1991

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