Functional characterization of the human 11 beta-hydroxysteroid dehydrogenase 1B (11 beta-HSD 1B) variant

Abstract

11 beta-Hydroxysteroid dehydrogenase type 1 (11 beta-HSD 1) catalyzes the interconversion of inactive into active glucocorticoids and has been shown to play a key role in metabolic disorders such as obesity and diabetes. 11 beta-HSD 1 belongs to the short chain dehydrogenases/reductases (SDR) and shares all common structural motifs typically for this protein superfamily. Unlike common SDRs, 11 beta-HSD 1 is N-terminally extended by a hydrophobic domain that anchors this enzyme in the endoplasmic reticulum (ER) membrane. Interestingly, the occurrence of 11 beta-HSD 1 transcripts lacking the N-terminal hydrophobic domain has repeatedly been reported in a variety of tissues, and the corresponding protein has been named 11 beta-HSD 1B. So far, no activity of 11 beta-HSD 1B has been observed, such that a physiological role could not be ascribed. In the present investigation, we showed for the first time that the truncated human 11 beta-HSD 1B form, expressed in the yeast Pichia pastoris, may indeed be active. However, this activity was prevented by the fact that 11 beta-HSD 1B is still kept attached to the ER membrane. Via computer assisted simulation and modeling, we identified a putative domain within the 11 beta-HSD 1 structure that could be responsible for this additional membrane attachment. By performing site-directed mutagenesis, heterologous expression, immunoblot analysis, and activity assays, we verified that this hydrophobic domain could indeed interact with the ER membrane and that some of the introduced mutations (V149R, V149E) led to a release of 11 beta-HSD 1B from membrane attachment without affecting its enzymatic activity. However, the activity of 11 beta-HSD 1B proved to be very unstable and was lost within hours after solubilization and release from the ER membrane. Importantly, 11 beta-HSD 1 constructs lacking the first 15 N-terminal amino acids and bearing additional amino acid substitutions (t15-V149R, t15-V149E) were then found to be soluble and to be stable in terms of enzyme activity. Combined, despite its occurrence in mammalian tissues, 11 beta-HSD 1B has obviously no physiological role since it is either inactive while being attached to the ER or it is rapidly losing activity once being released from intracellular membranes. Our findings with the t15-V149R and t15-V149E constructs are promising to further understand the complex mechanical and structural properties of 11 beta-HSD 1.