Effect of mutations of the human serpin protein corticosteroid-binding globulin on cortisol-binding, thermal and protease sensitivity.
Corticosteroid-binding globulin (CBG, transcortin) belongs to the serpin family of serine protease inhibitors (SERPINA6) and is mainly secreted by the liver. The negative acute phase protein CBG regulates free cortisol levels in the blood and distributes cortisol to its target tissues. So far no CBG serpin partner protease has been identified. However, its cleavage by human neutrophil elastase destroys ligand binding capacity and supposedly liberates cortisol at sites of inflammation. Here we report on the recombinant expression and secretion of human wild-type CBG and several novel mutants by human 293-EBNA cells. Functional characterization of wild-type and mutant CBG revealed distinct differences in ligand binding sensitivity to heat or elastase. Certain mutants are almost devoid of cortisol binding activity (Q232R and CBG Lyon), some display higher sensitivity for heat inactivation (G335V, Q232R and CBG Lyon) or for elastase cleavage (G335V). CBG mutant T342A is more resistant to elastase cleavage. Our data support the validity of the serpin structural concept. The expression system used provides functionally active human recombinant transcortin for further functional characterization of wild-type and human CBG mutant variants, which have been associated with altered serum free cortisol levels or pathophysiological constellations such as increased body weight, fatigue or hypotension.
Braun
Beate C
BC
Charité-Universitätsmedizin Berlin, Institut für Experimentelle Endokrinologie and EnForCé, Augustenburger Platz 1, D-13353 Berlin, Germany.
Meyer
Hellmuth-A
HA
Reetz
Antje
A
Fuhrmann
Ulrike
U
Köhrle
Josef
J
D002460:Cell Line; D006358:Hot Temperature; D006801:Humans; D006854:Hydrocortisone / Q000378:metabolism*; D007668:Kidney / Q000166:cytology; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics*; D014156:Transcortin / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; C534935:SERPINA6 protein, human; D014156:Transcortin; D012697:Serine Endopeptidases; D006854:Hydrocortisone