Merck

Naturally occurring mutations of human corticosteroid-binding globulin.

The Journal of clinical endocrinology and metabolism (2014-10-17)
Marc Simard, Lesley A Hill, John G Lewis, Geoffrey L Hammond
ABSTRACT

Corticosteroid-binding globulin (CBG) is encoded by SERPINA6. It is the major plasma binding protein of glucocorticoids and regulates plasma cortisol levels and bioavailability in humans. Several proteases target CBG and disrupt its steroid-binding properties. To date, most genetic deficiencies that alter plasma CBG levels or function have been identified in patients presenting with a variety of clinical conditions. The objective of the study was to test 32 previously uncharacterized nonsynonymous, single-nucleotide polymorphisms in SERPINA6 for their ability to alter CBG production and/or function. Human CBG mutants were produced in Chinese hamster ovary cells for ELISA, cortisol-binding activity measurements, and Western blotting as well as assays of their protease sensitivities. Eight naturally occurring CBG mutants with abnormal production and/or function were identified. Cortisol-binding affinity was markedly reduced for CBG H14Q and CBG H89Y, moderately decreased for CBG I279F, and undetectable for CBG R260L. By contrast, CBG H14R exhibited a decreased cortisol-binding capacity. Comparison of CBG levels in cell extracts and media by Western blotting revealed that CBG I48N and CBG P246Q have secretion defects. Two mutants (CBG I179V and CBG I279F) displayed reduced rates of cortisol-binding activity loss after exposure to three different proteases (neutrophil elastase, chymotrypsin, and LasB produced by Pseudomonas aeruginosa). Our data provide insight into how specific residues affect CBG secretion or function and illustrate the need to consider the various naturally occurring human CBG mutations in clinical evaluations of diseases associated with abnormalities in cortisol levels or activity.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium chloride solution, 0.85%
SAFC
Sodium chloride solution, 5 M
Sigma-Aldrich
Anti-SERPINA6 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Glycerin, meets USP testing specifications
Sigma-Aldrich
Sodium chloride solution, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥98.5% (GC)
Sigma-Aldrich
Glycerol solution, 83.5-89.5% (T)
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥99.0% (T)
Sigma-Aldrich
Sodium chloride solution, BioUltra, for molecular biology, ~5 M in H2O
Sigma-Aldrich
Sodium chloride, tested according to Ph. Eur.
Sigma-Aldrich
Sodium chloride, BioUltra, for molecular biology, ≥99.5% (AT)
Supelco
Sodium chloride, certified reference material for titrimetry, certified by BAM, ≥99.5%
Sigma-Aldrich
Sodium chloride, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
Sigma-Aldrich
Sodium chloride, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
Sodium chloride solution, 0.9% in water, BioXtra, suitable for cell culture
Sigma-Aldrich
Sodium chloride, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
DL-Glyceraldehyde 3-phosphate solution, 45-55 mg/mL in H2O
Supelco
Glycerin, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Sodium chloride-35Cl, 99 atom % 35Cl
Sigma-Aldrich
Glycerol solution, puriss. p.a., 86-89% (T)
USP
Glycerin, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Glycerol solution, puriss., meets analytical specification of Ph. Eur., BP, 84-88%
Sigma-Aldrich
Glycerol, FCC, FG
Sigma-Aldrich
Glycerol, ≥99.5%
Sigma-Aldrich
Glycerol, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for electrophoresis, ≥99% (GC)
Sigma-Aldrich
Glycerol, for molecular biology, ≥99.0%
Sigma-Aldrich
Glycerol, BioXtra, ≥99% (GC)
Sigma-Aldrich
Glycerol, tested according to Ph. Eur., anhydrous
Sigma-Aldrich
Glycerol, BioUltra, for molecular biology, anhydrous, ≥99.5% (GC)
Sigma-Aldrich
Glycerol, Vetec, reagent grade, 99%