Rabbit sex hormone binding globulin: primary structure, tissue expression, and structure/function analyses by expression in Escherichia coli.

Abstract

Sex hormone binding globulin (SHBG) is a homodimeric plasma protein found in mammals that binds sex steroids with high affinity and regulates their bioavailability. The protein is identical in structure and properties to the androgen binding protein (ABP) found in the male reproductive tract. We have isolated a 1245-base pair rabbit SHBG cDNA encoding a reading frame for a signal peptide followed by a protein of 367 amino acids, which shares 79.0, 68.1 and 63.2% amino acid identity with the corresponding human, rat and mouse proteins respectively. Northern blot and hot-nested PCR analyses indicated that rabbit SHBG is produced from a 1.6 kilobase mRNA in the liver of both sexes and in the testis. The rabbit SHBG cDNA was inserted into pGEX-1 lambda T for expression of a glutathione S-transferase/SHBG fusion protein in Escherichia coli. The bacterial product bound 5 alpha-dihydrotestosterone (DHT) in the same manner as the corresponding protein in serum. The dissociation constants (Kd) for rabbit and human SHBGs produced in E. coli were 11.1 +/- 1.1 nM and 2.1 +/- 0.6 nM respectively, and rabbit SHBG formed a less stable protein-steroid complex (t1/2 = 5 min) than human SHBG (t1/2 > 60 min). Unlike human SHBG, rabbit SHBG does not bind estradiol with high affinity. To aid in the identification of differences in the sequences of rabbit and human SHBG, which determine species differences in steroid-binding affinity and specificity, chimeras containing the 5'-terminal half of SHBG from one species and 3'-terminal half of SHBG from the other species were constructed and expressed. It was found that the chimeric proteins assumed similar steroid-binding affinity and specificity as the wild-type proteins when the amino (N)-terminal half of SHBG was derived from the same species. Replacement of the carboxyl (C)-terminal half of rabbit SHBG by the corresponding region of the human molecule increased the integrity of its steroid-protein complex. This supports the concept that amino acids within the N-terminal half of SHBG constitute the steroid-binding domain while the C-terminal half of the molecule may provide structural stability to the protein and its steroid-binding site.