Sex hormone-binding globulin (SHBG) is a homodimeric glycoprotein that transports sex steroids in the blood and extravascular fluids, such as seminiferous tubule fluid, where it is often referred to as androgen-binding protein (ABP). A cDNA for the human SHBG precursor polypeptide was expressed in mammalian cell lines. Recombinant human SHBG bound steroids with the same affinity and specificity as natural SHBG, and was immunologically indistinguishable from its natural counterpart. Site-directed mutagenesis of the SHBG cDNA was used to introduce amino acid substitutions that selectively removed glycosylation sites from SHBG. Analysis of these glycosylation mutants demonstrated that carbohydrates are not involved in steroid binding, but the lack of both N-linked oligosaccharides reduced the level of SHBG secretion and/or production from CHO cells. In addition, the subunit size heterogeneity associated with SHBG is due to the differential utilization of the two consensus sites for N-glycosylation, and that subunits that comprise a given dimer are glycosylated and processed in the same way. An additional N-glycosylation site associated with an electrophoretic variant of SHBG was shown to be utilized and found not to affect steroid binding. Analyses of human SHBG/rat ABP chimeras and human SHBG C-terminal truncation mutants expressed in E. coli revealed that the steroid-binding domain is located within the N-terminal 205 amino acids of human SHBG. Specific amino acid substitutions in human SHBG produced mutants with altered steroid-binding specificity and demonstrated that Lys{dollar}\sp{lcub}134{rcub}{dollar}-Met{dollar}\sp{lcub}139{rcub}{dollar} interact with the A/B ring structures of steroids, and residues in a more N-terminal location may also contact steroid ligands. Human SHBG mutants with substitutions at residues Ile{dollar}\sp{lcub}138{rcub}{dollar}-Phe{dollar}\sp{lcub}148{rcub}{dollar} are defective in their ability to dimerize, especially in the absence of steroid and/or divalent cations, but readdition of these agents restores dimer formation. These data have led us to conclude that SHBG is a modular protein comprising an N-terminal steroid-binding and dimerization domain, and a C-terminal domain(s), which contains a phylogenetically conserved N-glycosylation site that may be required for other activities, such as recognition by a plasma membrane receptor
