A Leu----His substitution at residue 93 in human corticosteroid binding globulin results in reduced affinity for cortisol.
A steroid binding capacity assay and a radioimmunoassay were both used to measure corticosteroid binding globulin (CBG) in serum samples from 22 patients with sepsis. An approximately 50% discordancy between the two values in one patient suggested the presence of a CBG variant with reduced affinity for cortisol, and this was confirmed by Scatchard analysis. We therefore used the polymerase chain reaction to amplify exons that encode for human CBG from the genomic DNA of this patient. This revealed two mutations within the coding sequences: one of which results in a Leu----His substitution at residue 93 and another which encodes a Ser----Ala substitution at residue 224 of the human CBG polypeptide. To assess the impact of each substitution on the steroid binding affinity of CBG, each mutation was introduced separately into a normal human CBG cDNA, and the normal and mutated cDNAs were expressed in Chinese hamster ovary cells. Scatchard analysis of the CBG produced in culture indicated that the His93 mutation (Kd = 2.24 +/- 1.75 nM) reduced the cortisol binding affinity of CBG (mean +/- SD) significantly (P less than 0.024) when compared to normal CBG (Kd = 0.64 +/- 0.31 nM), while the Ala224 mutation (Kd = 0.63 +/- 0.33 nM) did not influence cortisol binding affinity. We therefore conclude that residue 93 may play an important role in determining the structure of the CBG steroid binding site.
Smith
C L
CL
Department of Obstetrics and Gynecology, University of Western Ontario, Victoria Hospital, London, Canada.
Power
S G
SG
Hammond
G L
GL
D000368:Aged; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA; D005260:Female; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D006854:Hydrocortisone / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D014156:Transcortin / Q000235:genetics / Q000378:metabolism*
D006639:Histidine; D004247:DNA; D014156:Transcortin; D007930:Leucine; D006854:Hydrocortisone