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

Anne Mette Frøbert, Malene Brohus, Julia N C Toews, Phillip Round, Ole Fröbert, Geoffrey L Hammond, Michael T Overgaard

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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 ± 0.04 nm for human and 1.32 ± 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.

Original languageEnglish
JournalFEBS open bio
Volume12
Issue number2
Pages (from-to)362-378
Number of pages17
ISSN2211-5463
DOIs
Publication statusPublished - Feb 2022

Bibliographical note

Funding Information:
Brown bear liver tissue from a euthanized bear was kindly provided by Orsa Rovdjurspark in Sweden. The authors thank Helene Halkjær Jensen, Christian Holt, Rasmus Wollenberg, and Teis Søndergaard for excellent laboratory technical assistance. This work was supported by a PhD grant R286‐2018‐367 (to AMF) and grant R126‐2012‐12408 (to OF and MTO) from The Lundbeck Foundation.

Publisher Copyright:
© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies

Keywords

  • Animals
  • Dihydrotestosterone/metabolism
  • Humans
  • Recombinant Proteins
  • Sex Hormone-Binding Globulin/chemistry
  • Steroids/metabolism
  • Ursidae

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