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Functional characterization of sex hormone-binding globulin genetic polymorphism Wu, Tsung-Sheng


Human plasma SHBG is produced by the liver, and it transports biologically active sex steroids and determines their availability to target tissues. The 4.3 kb human SHBG transcriptional unit encoding a signal polypeptide for secretion followed by two laminin G (LG)-like domains is utilized by hepatocytes. The N-terminal LG domain of SHBG contains a region responsible for homodimer formation, and a steroid ligand-binding site that accommodates androgens and estrogens in opposite orientations. Among over 250 genetic polymorphisms identified in human SHBG, few are functionally characterized. In this research, I have performed a comprehensive analysis of functionally relevant SHBG single nucleotide polymorphisms (SNPs). Nine out of nineteen non-synonymous SNPs within the coding region of SHBG N-terminal LG domain were shown to encode SHBG mutants with abnormal properties in steroid ligand binding, calcium coordination, fibulin-2 interaction, glycosylation or secretion. In particular, SHBG R123H (encoded by rs143269613) has a general reduction in affinity for steroid ligands, whereas SHBG E176K (encoded by rs372114420) has a higher affinity specifically for estradiol. Crystallography revealed that instead of losing the structural integrity of the steroid-binding site, reduced flexibility of the loop region that covers the steroid-binding site, and conformational changes at the opening rim of a putative estradiol entrance, likely account for the abnormal steroid-binding affinities of SHBG R123H and SHBG E176K, respectively. Among eight SNPs within SHBG regulatory sequences selected for analysis, only rs138097069 increases SHBG promoter activity. In silico prediction revealed that rs138097069 is located within a putative FXR binding site, while rs6257, which is linked to low plasma SHBG concentrations, is located within a putative FOXA2 binding element. In HepG2 cells, GW4064-activated FXR and overexpressed FOXA2 both suppress SHBG expression by direct binding to their corresponding binding elements in an HNF4⍺-independent manner. By contrast, knock-down of FXR reduces, while knock-down of FOXA2 induces, HNF4⍺ expression and SHBG production. Characterization of functional SHBG SNPs has provided molecular explanations of how genetic differences contribute to SHBG production and function, and has identified possible roles for two novel regulators, FXR and FOXA2, in a more complex regulatory network that determines SHBG expression.

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