Characterization and comparison of recombinant full‐length ursine and human sex hormone‐binding globulin

Abstract

Sex hormone-binding globulin (SHBG) regulates the bioavailability of sex steroid hormones in the blood. Levels of SHBG increase markedly in brown bears (Ursus arctos) during hibernation, suggesting that a key regulatory role of this protein is to quench sex steroid bioavailability in hibernation physiology. To enable characterization of ursine SHBG and a cross species comparison, we established an insect cell-based expression system for recombinant full-length ursine and human SHBG. Compared with human SHBG, we observed markedly lower secretion levels of ursine SHBG, resulting in a 10-fold difference in purified protein yield. Both human and ursine recombinant SHBG appeared as dimeric proteins in solution, with a single unfolding temperature of ~58 °C. The thermal stability of ursine and human SHBG increased 5.4 and 9.5 °C, respectively, in the presence of dihydrotestosterone (DHT), suggesting a difference in affinity. The dissociation constants for [ 3 H]DHT were determined to 0.21 AE 0.04 nM for human and 1.32 AE 0.10 nM for ursine SHBG, confirming a lower affinity of ursine SHBG. A similarly reduced affinity, determined from competitive steroid binding, was observed for most steroids. Overall, we found that ursine SHBG had similar characteristics to human SHBG, specifically, being a homodimeric glycoprotein capable of binding steroids with high affinity. Therefore, ursine SHBG likely has similar biological functions to those known for human SHBG. The determined properties of ursine SHBG will contribute to elucidating its potential regulatory role in hibernation physiology.Sex hormone-binding globulin (SHBG) is a plasma protein that transports and regulates the bioavailability of sex steroids in the blood by adjusting the equilibrium between free and protein-bound steroid [1]. Steroids are lipophilic with low solubility in water and are therefore bound to various binding proteins when transported in the circulation [2]. Sex steroids, including androgens and estrogens, are important regulatory molecules that control sexual maturation and reproduction as well as regulate other sexually dimorphic Abbreviations AEX, anion exchange chromatography; DCC, dextran-coated charcoal; DHT, 5α-dihydrotestosterone; MALDI-ToF MS, matrix-assisted laser desorption ionization-time of flight mass spectrometry; pFBD, pFastBac TM Dual; RBA, relative binding affinity; SEC-MALS, size exclusion chromatography-multi-angle light scattering; SHBG, sex hormone-binding globulin.