The fascinating complexities of steroid-binding enzymes

Enzymes that modulate the level of circulating steroid hormone can be used to combat steroid-dependent disorders. Members of the NADPH-dependent short chain dehydrogenase/reductase (SDR) family control blood pressure, fertility, and natural and neoplastic growth. Despite the fact that only one amino acid residue is strictly conserved in the 60 known members of the family, all appear to have the dinucleotide-binding Rossmann fold and homologous catalytic residues containing the conserved tyrosine. Variation in the amino acid composition of the substrate binding pocket creates specificity of binding for steroids, prostaglandins, sugars and alcohols. Licorice induces high blood pressure by inhibiting an SDR in the kidney, and appears to combat ulcers by inhibiting another in the stomach. Detailed X-ray analyses of various members of the family should allow the design of potent, tissue-specific, highly selective inhibitors.     

A proposed consensus steroid-binding sequence--a reply

A consensus sequence has been proposed as necessary for steroid binding on the basis of sequence homology in steroidogenic enzymes, steroid binding proteins and steroid receptors. We have mapped the limit of sequences important for steroid binding in the mouse estrogen receptor using Scatchard analysis of deletion mutants. Since a mutant lacking the entire proposed consensus is still able to bind estradiol with essentially wild type affinity, the functional significance of this motif is unclear.     

Localization of the steroid-binding site of the human sex steroid-binding protein of plasma (SBP or SHBG) by site-directed mutagenesis.

The amino-terminal region of the human sex steroid-binding protein of plasma (SBP or SHBG) containing K134 and M139 was found to represent part of the steroid-binding site. This was accomplished by constructing and expressing site-directed mutants having the following replacements: M139L, M139K, M139S, K134A, H235S, and Y57F. The results indicated that M139L and H235S were fully-active, K134A and Y57F were 50 and 67% active, M139K was 7% active, and M139S was inactive. These results support affinity-labeling data indicating that both K134 and M139 are located in or near the site, and suggest that Y57 may play a role in steroid binding. The fully active H235S mutant reveals that H235 is not involved in the steroid-binding process.     

Purification of the steroid-binding core of porcine estrogen receptor

The steroid-binding core of estradiol receptor was purified from pig uterus cytosol by a protocol consisting of (1) adsorption to heparin-sepharose, (2) enzymatic release of the receptor core, (3) DEAE-chromatography, (4) Sephadex G-150 filtration and (5) chromatography on heparin-sepharose. The final product was approximately 18000-fold enriched over the starting material. It consisted of at least 18% core protein resembling dimeric microsomal receptor with a molecular mass of 75 kDa and an isoelectric point of 5.8 (microheterogeneity). A goat antiserum raised against the preparation contains immunoglobulins G precipitating estradiol-receptor complexes, and antibodies releasing the steroid from its binding site.     

Isolation of steroid-binding proteins on new affinity adsorbents]

Steroid-binding proteins of human blood are extracted and investigated on corticosteroid-hydrazide-oxypropyl-adsorbents by the affinity chromatography. The role of structural changes in the matrix-spacer-ligand system for different types of adsorbents is studied as well as a dependence of sorption on pH and salt environment. A dependence of the protein sorption on the isolated hydrazide groups is revealed. It is shown that hydrocortisone displays the highest binding activity to the protein pool in the affinity electroimmunoassay while the substance S-Reichstein and its 16-alpha-methyl analog--the least one.     

Receptor for sex steroid-binding protein of endometrium membranes: solubilization, partial characterization, and role of estradiol in steroid-binding protein-soluble receptor interaction.

The sex steroid-binding protein (SBP) receptor was solubilized from the membranes of human premenopausal endometrium with the zwitterionic detergent CHAPS. The binding activity of the soluble receptor was studied, allowing it to interact with [125I]SBP and precipitating the complex with polyethylene glycol 8,000. The interaction of SBP with the soluble receptor was specific, saturable, and at high affinity. Indeed, the specific binding was definitely improved on the solubilized form of the receptor. The effect exerted by sex steroids on the interaction of SBP with receptor was also examined on both the soluble and membrane-bound forms. At physiologic doses (10(-8) M) estradiol inhibits the binding at a significant extent on the soluble receptor, but not on membrane-bound form. The dose of estradiol required to significantly inhibit the SBP-specific binding was dependent on the form of receptor. In membrane-bound receptor the inhibiting dose of estradiol was higher than its physiologic concentration. Thus, it is likely that, while soluble receptor cannot recognize the complex steroid-SBP, membrane-bound receptor can interact both with "unliganded" SBP and with the estradiol-SBP complex (but not with androgen-SBP complexes) in an estrogen-dependent tissue like human endometrium.     

Complete enzymatic deglycosylation of native sex steroid-binding protein (SBP or SHBG) of human and rabbit plasma: effect on the steroid-binding activity.

An enzymatic procedure for the complete removal of the N-linked and O-linked oligosaccharide side chains of the sex steroid-binding proteins (SBP or SHBG) of human and rabbit plasma under native conditions is described. Deglycosylation was catalyzed by N-glycanase, neuraminidase, and O-glycanase and was monitored by SDS-PAGE, lectin blotting, and molecular weight analyses by electrospray mass spectrometry. Digestion of rabbit SBP with N-glycanase generated a major 39,777-Da protein and two minor ones of 39,389 and 39,545 Da. The molecular weight of the major protein agrees with the molecular weight calculated from the sequence of the sugar-free polypeptide monomer (39,769 Da: Griffin, P.R., Kumar, S., Shabanowitz, J., Charbonneau, H., Namkung, P.C., Walsh, K.A., Hunt, D.F., & Petra, P.H., 1989, J. Biol. Chem. 264, 19066-19075), whereas the other two are deglycosylated proteolytic cleavage products lacking the TQR and TQ sequences at the amino-terminus. The N- and O-linked side chains of human SBP were removed by sequential digestion with N-glycanase and neuraminidase/O-glycanase. A 38,771-Da protein was generated, which agrees well with the molecular weight of the sugar-free polypeptide monomer (Walsh, K.A., Titani, K., Kumar, S., Hayes, R., & Petra, P.H., 1986, Biochemistry 25, 7584-7590). N-deglycosylation of human and rabbit SBP has no effect on the steroid-binding activity, but removal of the O-linked side chains of N-deglycosylated human SBP results in an apparent 50% loss of steroid-binding activity and an increase in the Kd for the binding of 5 alpha-dihydrotestosterone from 0.3 mM to 0.9 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative study of primates' transcortin: immunoreactivity and steroid-binding activity

To investigate the phylogenic aspect of transcortin (corticosteroid-binding globulin, CBG), the immunoreactivity of transcortin with anti-human transcortin antiserum was studied in primates. The anti-human transcortin antibody was recognized by plasma proteins obtained from Catarrhini, taxonomically the most evolved monkey group. The immunoreactivity was not observed in plasma obtained from Platyrrhini and Prosimiae, classified as less evolved monkey groups than Catarrhini. Though comparison of immunoreactivity among different classes of Catarrhini was difficult because of non-parallelism of their displacement curves, displacement of 125I-labelled human transcortin from the antiserum by 1:10 and 1:100 diluted plasma was highest in human followed by Pongidae, Cercopithecoidea. The immunoreactivity of thyroxine-binding globulin (TBG) with anti-human TBG antiserum was also examined. The anti-human TBG antibody was only recognized in plasma from Pan (anthropoid ape) among Pongidae, highly evolved monkeys among Catarrhini. The existence of immunoreactive transcortin and TBG to respective human protein antibody in the highly evolved ape agreed well with the cladogenetic division of primate species delineated by Goodman and Moore (1971). Cortisol-binding activity of transcortin was detected in all monkeys except three, tafted capuchin monkey, night monkey and cotton-headed tamarin, which belong to Platyrrhini. The absence of cortisol-binding activity in these animals might be attributed to high levels of endogenous cortisol and low cortisol-binding capacity of transcortin. It is speculated that the structure of the immunoreactive site in transcortin could be modified by evolution without affecting the biologically important site, the site for cortisol binding.     

Prostatic steroid-binding protein. Isolation and characterization of C3 genes

Prostatic steroid-binding protein, whose expression is stimulated by androgens, consists of two subunits, one containing the polypeptides C1 and C3 and the other containing the polypeptides C2 and C3. We have isolated and sequenced cDNA clones specific for C3 mRNA and used them to isolate and characterize genomic clones for two C3 genes. Both genes are 3.2 kilobases with identical exon/intron arrangements, which is similar to the organization of the C1 and C2 genes, suggesting that they may have arisen by duplications of an ancestral gene. Finally, homologous human genes have not been detected.     

Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma

We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4°C with high affinity (K _a = 1.49 ± 0.09 × 10⁹ M⁻¹; 0.17 ± 0.02 × 10⁷ M⁻¹) and low binding capacity (B _max = 3.24 ± 0.84 × 10⁻⁵ M; 0.33 ± 0.06 × 10⁻⁴ M). The binding affinity and capacity of testosterone at 23 and 36°C, respectively were similar to those determined at 4°C. However, oestradiol showed no binding activity at 36°C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36°C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.     

Direct evidence for the localization of the steroid-binding site of the plasma sex steroid-binding protein (SBP or SHBG) at the interface between the subunits.

Complete dissociation of dimeric plasma sex steroid-binding protein (SBP or SHBG) was obtained in 6 M urea at 10 degrees C. Removal of urea resulted in the refolding of monomers, followed by reformation of dimeric SBP, which migrates with the same mobility as the native protein. Dimerization does not require Ca+2 or steroid. Renatured monomers yield dimers with dissociation constants for 5 alpha-dihydrotesterone (DHT) and 17 beta-estradiol (E2) indistinguishable from those of native human SBP. This phenomenon was also demonstrated by mixing human and rabbit SBPs that, upon renaturation, form a hybrid dimer composed of one human subunit and one rabbit subunit. The hybrid binds both DHT and E2 in contrast to rSBP, which only binds the androgen. Therefore, we conclude that (1) docking of the two subunits creates an asymmetric steroid-binding site located at the interface between the subunits, and (2) only one face of the dimer defines the specificity for binding E2 by encompassing portion of a structural motif that recognizes the flat ring A of E2. The remaining portion, which recognizes the saturated ring A of DHT, is shared by both faces of the dimer. Because native monomers do not exist alone, the often-asked question of whether the SBP monomer binds steroid can be considered meaningless; steroid-binding activity is expressed only in the dimeric state. Finally, formation of the hybrid indicates that SBP dimerization represents a conserved event during the molecular evolution of SBP, suggesting that the structural elements responsible for dimerization will be homologous in SBPs from other species.     

Properties of sex steroid-binding protein from Xenopus laevis blood serum and detection of an estradiol-binding component in frog liver cytosol which differs from sex steroid-binding protein]

Binding and physico-chemical properties of sex steroid-binding protein (SBP) from blood serum and those of estrogen-binding components from liver cytosol of pubertal male and female species of clawed frog Xenopus laevis were studied. It was shown that SBP from both sex species of X. laevis specifically binds estradiol (E2) (Ka=5 . 10(6) M-1). Concentration of SBP binding sites for E2 is 7 . 10(-12) mole per mg of protein. Testosterone 5alpha-dihydrotestosterone and E2 effectively compete with [3H]-E2 for SBP binding sites. Hexestrol, progesterone and corticosterone are weak competitors; estrone and E2-17-hemisuccinate do not compete at all. The Strokes radius of SBP is 4.4 nm; sedimentation coefficient is 4.6S. Molecular weight of SBP is 88000; f/f0 is 1.5 SBP from male frog sera has been purified 8.6-fold with 13% yield. Gel-filtration of [3H]-E2 complexes with liver cytosol proteins shows that the livers of male and female frog X. laevis consol proteins shows that the livers of male and female frog X. laevis contain very low amounts of macromolecular component, which specifically binds E2; this component differs from serum SBP in size and in hormonal specificity. It is assumed that this component is a receptor for estrogens.