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.     

Immunohistochemical detection of the sex steroid-binding plasma protein in human mammary carcinoma cells

A sex steroid-binding plasma protein-like antigen has been detected in human mammary carcinoma cells. A monospecific antiserum was used, and this protein was located mainly on the cytoplasmic membranes. These results are in agreement with a recent hypothesis according to which steroid hormones could be carried into cells by specific binding plasma proteins.     

Characterization of a cDNA coding for sex steroid-binding protein of human plasma

A cDNA (912 nucleotides) coding for human plasma sex steroid-binding protein (SBP) was characterized from a phage clone previously isolated by screening a Charon 21A human liver cDNA library with rat androgen binding protein (ABP) cDNA. The deduced amino acid sequence from the cDNA indicated that the insert was a partial clone coding for 281 amino acids starting with residue 92 (glycine) encompassing the alternating leucyl residues and the carboxyl-end 373 (histidine) as previously reported [(1986) Biochemistry 25, 7584]. The potential polyadenylation signal sequence ATTAAA is present as part of the 3'-coding region and the stop codon TAA. Both are followed by a short 20 untranslated nucleotides and a poly(A) tract of 49 nucleotides. Significant homologous sequences (76%) at the DNA level exist between human SBP and rat ABP which might suggest the possibility that both evolved from a common primordial gene. Demonstration of the presence of an SBP cDNA in a human liver cDNA library provides the first evidence that liver is the site of SBP biosynthesis.     

Resolution of a disordered region at the entrance of the human sex hormone-binding globulin steroid-binding site

The crystal structure of human sex hormone-binding globulin (SHBG) has revealed how 5alpha-dihydrotestosterone intercalates between the two seven-stranded beta-sheets of its amino-terminal laminin G-like domain. However, a region of disorder (residues 130 to 135 of SHBG) was identified together with a zinc-binding site in immediate proximity to the steroid. It has been important to resolve the structure of this region because previous studies have suggested that these residues may contribute to steroid binding directly. Here, we present the 2.35 A and 1.7 A crystal structures of the amino-terminal LG domain of SHBG obtained from a tetragonal crystal form and by EDTA-soaking of a trigonal crystal form, respectively. In both of these new structures, residues Pro130 to Arg135 are now clearly visible. Substitution of the two residues (Leu131Gly and Lys134Ala) pointing towards the steroid has shown that only Leu131 contributes significantly to steroid binding. Rather than covering the steroid-binding pocket in an extended conformation, a 3(10) helical turn is formed by residues Leu131 to Lys134 in this segment. Unfolding of this secondary structure element can either facilitate the entry of the steroids into the binding site or modulate the important contribution that Leu131 makes to steroid binding. A comparison with previous structures supports the concept that zinc binding re-orients the side-chain of His136, and this residue serves as a lever causing disorder within the loop structure between Pro130 and Arg135.     

Purification of the sex steroid-binding protein from common carp (Cyprinus carpio) plasma.

1. Sex steroid-binding protein was purified from common carp plasma. 2. Testosterone- and estradiol-binding activity existed at the same fraction eluted from gel Sepharose CL-2B, DEAE-Sephacel, hydroxylapatite and HPLC. 3. The molecular weight of the sex steroid-binding protein was 194,000. 4. At 50% displacement the order in which the steroids displaced [3H]testosterone bound to the binding protein was as follows: androstenedione greater than estradiol-17 beta greater than 11-deoxy-17-hydroxycorticosterone greater than 17 alpha-hydroxyprogesterone greater than progesterone greater than deoxycorticosterone greater than estrone greater than 11-ketotestosterone greater than 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one greater than androstenedione greater than pregnenolone greater than cortisone greater than cortisol.     

Purification and characterization of the sex steroid-binding protein of rabbit serum. Comparison with the human protein

The sex steroid-binding protein (rSBP) of immature rabbit serum was purified to homogeneity by the sequential use of DEAE-cellulose chromatography, affinity chromatography on 5alpha-dihydrotestosterone-17 beta-succinyl-diaminoethyl-(1,4-butanediol diglycidyl ether)-agarose, agarose (Bio-Gel-A-0.5m) gel filtration, and preparative polyacrylamide gel electrophoresis. The cumulative yield is 13%. Homogeneity of rSBP was shown by the equilibrium sedimentation ultracentrifugation in 6 M guanidine HCl containing 0.1 M mercaptoethanol which yields an average molecular weight of 36,475 +/- 865. Analytical gel electrophoresis in the presence of sodium dodecyl sulfate and gel filtration on agarose yield a molecular weight of 57,000 and 120,000, respectively. The variation is due to a 30% carbohydrate content. The amino acid composition is reported. Comparison of the rabbit and human SBP indicate that they are different in both their molecular and functional properties.     

Modification in the purification of the sex steroid-binding protein of human serum by affinity chromatography

A purification procedure for the sex steroid-binding protein of human serum is described. The procedure is significantly superior to that recently published (K. E. Mickelson, D. C. Teller, and P. H. Pétra, 1978, Biochemistry17, 1409–1415) and should replace it for the routine preparation of homogeneous protein in relatively larger quantities. The steps involved diethylaminoethyl-cellulose chromatography, affinity chromatography on 5α-dihydrotestosterone-17α-hexanyldiaminoethyl-(1,4-butanediol diglycidyl ether)-agarose, and preparative polyacrylamide gel electrophoresis. The most important difference between this new procedure and that previously published is the affinity adsorbent with contains the steroid covalently linked at the 17α-position rather than the 17β-position. This modification allows the purification of at least 12 mg of homogeneous protein per preparation with a 63% total yield. The properties of the homogeneous protein are the same as previously described.     

Interaction of blood sex steroid-binding globulin-steroid complexes with plasma membranes of human decidual endometrium cells

Plasma membranes of decidual tissue cells specifically bind the sex steroid-binding globulin (SBP) complexes with estrogen (estradiol, estriol, estrone) and with the pharmacological agent danazol but do not interact with the SBP-testosterone or SBP-dihydrotestosterone complexes. The selectivity of interaction of the SBP-steroid complexes with decidual tissue cellular membranes provide evidence for the active role of SBP in the realization of steroid effects on the target tissue.     

Mammalian expression of the human sex steroid-binding protein of plasma (SBP or SHBG) and testis (ABP). Characterization of the recombinant protein.

A full-length 1,209 bp cDNA encoding the human sex steroid-binding protein of plasma (SBP or SHBG) and testis (ABP) was constructed and expressed in BHK-21 cells. The sequence agrees with the published gene and protein sequences. The cells were found to secrete SBP following transfection and G418r selection. The recombinant protein binds 5 alpha-dihydrotestosterone with a Kd of 0.28 nM. It also binds testosterone and 17 beta-estradiol but not progesterone, estrone or cortisol revealing a steroid-binding specificity identical to that of human SBP. SDS-PAGE patterns are less complex than human SBP and show a monomeric molecular weight of about 43 kDa.     

A new method for measurements of plasma protein steroid-binding kinetics in human plasma at 37 C

The equilibrium kinetics in vivo of free and protein-bound steroid hormone were examined at 37 C. Human plasma was pumped through a 1 m PVC catheter and tritiated steroid hormone continuously added at the inlet. The plasma was collected and rerun through a new catheter 24 h later. A significantly (P less than 0.05) higher uptake of steroid hormone was observed in the first parts of the catheter during the first passage when compared to the second passage. The results indicate that non-equilibrium conditions existed for testosterone (greater than 10 sec), estradiol (4 sec), estrone (greater than 2 sec), and estriol (greater than 2 sec) while no delay was observed for progesterone. The results indicate that the counter-current transfer between the testicular and ovarian vessels, respectively, may create a physiologically important, temporarily increased concentration of available hormones in the arterial blood supply to the organs.     

Binding of xenoestrogens to the sex steroid-binding protein in plasma from Arctic charr (Salvelinus alpinus L.)

A specific sex steroid-binding protein (SBP) is believed to be involved in regulation of circulating sex steroids, steroid delivery to target cells and intracellular signalling in sex steroid-sensitive tissues. In the present work, interactions between xenoestrogens and the plasma SBP in Arctic charr (Salvelinus alpinus L.) were determined using ligand-protein binding studies. The test compounds were all able to displace tritiated 17 beta-estradiol (E2) from the Arctic charr SBP (acSBP) in a competitive and dose-dependent manner. The acSBP affinities for the xenoestrogens ranged over several orders of magnitude (17 beta-estradiol>ethynylestradiol (EE2)>zearalenone (ZEA)>diethylstilbestrol (DES)>genistein (GEN)>bisphenol A (BPA), 4-t-octylphenol (OP)>o,p'-DDT, and dieldrin (DIN)), but were consistently lower than that of 17 beta-estradiol (about 4 x 10(2) -10(6)-fold less potent). The relative binding affinity (RBA) for selected chemicals were independent of both gender, age and maturation status, as well as variations of acSBP binding affinity. The affinity of endogenous steroids and estrogen mimics for the acSBP shows a high correlation to the affinity for the rainbow trout SBP, thus suggesting a phylogenetically conserved ligand-binding site between closely related species. Furthermore, it is argued that interaction with the acSBP- and SBP-mediated processes may introduce novel pathways for endocrine disruption, which may work in concert with the classical receptor-mediated effects.     

The amino acid sequence of the sex steroid-binding protein of rabbit serum

The amino acid sequence of the sex steroid-binding protein (SBP or SHBG) of rabbit serum, specific for binding testosterone and 5 alpha-dihydrotestosterone, was determined using a complementary combination of mass spectrometric and Edman degradation techniques. The monomeric unit of the homodimeric protein is a single chain glycopeptide of 367 amino acid residues, with N-linked oligosaccharide side chains at Asn-345 and Asn-361 and disulfide bonds connecting Cys-158 to Cys-182 and Cys-327 to Cys-355. The polypeptide molecular weight of the monomer calculated from the sequence is 39,769. The molecular weight of the homodimer including 9% carbohydrate is 87,404. The sequence contains a relatively hydrophobic segment between Trp-241 and Leu-282, which includes many leucine residues in an alternating pattern. An amino acid sequence repeat is also located within that segment. Both of these patterns are present in human SBP and in the androgen-binding protein of rat epididymis. The sequence data indicate that the previously reported microheterogeneity of rabbit SBP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis reflects variants generated by differential glycosylation of the monomer rather than different gene products. Seventy-nine percent of the amino acids of rabbit SBP are identical to those of human SBP; rabbit SBP thus joins human SBP and rat androgen-binding protein in one gene family that is distinct from the steroid hormone receptor superfamily. It appears that the problem of binding sex steroid hormones has been solved independently in two different gene families that contain completely different steroid-binding domains. Since the nonhomologous steroid-binding domains of both families of proteins recognize essentially the same steroid structure, it will be interesting to determine the structural basis of the two different protein designs that lead to similar steroid-binding specificity.     

Purification and physico-chemical properties of the sex steroid-binding globulin of human blood

A new technique for purification of the human sex hormone-binding globulin (SHBG) is described. This technique includes affinity chromatography of blood serum on cortisol-Sepharose, (NH4)2SO4 fractionation, gel filtration on a Bio-Gel P-300 column and chromatography on a concanavalin A-Sepharose 4B column. From 21 of retroplacental serum 10 mg of pure SHBG (25% yield) has been obtained. Upon gel filtration SHBG behaved as a biopolymer with Mr of 120,000. The molecular weight of SHBG as determined by electrophoresis was shown to be equal to 50,000. SHBG has a sedimentation constant of s20, w of 4.7S, pI of 5.75, extinction coefficient A1%(280,1cm) = 10,5 and association constants of 4.5 X 10(8) and 3.5 X 10(6) M-1 for 5 alpha-dihydrotestosterone and cortisol, respectively. The amino acid and carbohydrate contents of SHBG were determined.     

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.     

The plasma membrane of epididymal epithelial cells has a specific receptor which binds to androgen-binding protein and sex steroid-binding protein.

The binding of [3H] delta 6-testosterone photoaffinity-labelled rat androgen-binding protein (rABP) has been studied in an enriched fraction of plasma membranes of epithelial epididymal cells in immature (15 days) and adult rats (40 days). The binding was maximal in less than 30 min and more rapid at 4 degrees C than at 34 degrees C. It was calcium and pH dependent. Scatchard plots of the binding data gave curvilinear plots with two types of binding sites corresponding to a K(ass1) of 18.2 nM-1 and K(ass2) of 1.6 nM-1 (2.2 x 10(11) sites/mg protein and 5.4 x 10(11) sites/mg protein, respectively). In adult rats, only one type of binding site was found, with a K(ass) of 3.7 nM-1 (4.5 x 10(11) sites/mg protein). The number of receptors was 5-fold lower in the cauda than in the caput of the epididymis. The pretreatment of the isolated intact cells with streptozotocin induced a 45% reduction of the binding. Only unlabelled rABP and hSBP (human sex steroid-binding protein) but not other proteins (lactotransferrin, serotransferrin, asialofetuine, fetuine and bovine serum albumin) competed with the labelled ligand to bind plasma membranes. The membrane fraction was solubilized by triton X-100. Its incubation with labelled rABP and hSBP provoked the elution of the tracer as an aggregate into the void volume fraction of superose 6B mini-gel filtration columns. Structural homology between hSBP and rABP could be responsible for the common behaviour of the steroid-carrier molecules for the ABP receptor of rat epididymal epithelial cells.     

Steroid-binding site of human and rabbit sex steroid binding protein of plasma: fluorescence characterization with equilenin

The interaction of the estrogen d-3-hydroxy-1,3,5(10),6,8-estrapentaen-17-one (equilenin) with the human and rabbit sex steroid binding proteins (hSBP and rSBP, respectively) has been investigated by using fluorescence and absorption spectroscopy. Equilenin competes for the binding of 5 alpha-dihydrotestosterone. The calculated binding constant of equilenin for rSBP is 1.9 X 10(7) M-1 at 4 degrees C, which can be compared with the binding constant of 5.7 X 10(7) M-1 reported for hSBP [Ross, J.B.A., Torres, R., & Petra, P.H. (1982) FEBS Lett. 149, 240]. The results of fluorescence quenching experiments with the collisional quenchers KI and acrylamide indicate that the bound steroid has limited accessibility to the bulk solvent and that there are no anionic surface groups near the steroid-binding site. The fluorescence excitation spectra of SBP-equilenin complexes are similar to the absorption spectra of equilenin in low-dielectric solvents. The fluorescence emission of the SBP-equilenin complexes, however, exhibits wavelength shifts (red shifts) opposite to those of the steroid in low-dielectric solvents or complexed with beta-cyclodextrin (blue shifts) but similar to the red shift produced by addition of the proton acceptor triethylamine to equilenin in cyclohexane. These data indicate that the steroid-binding site of hSBP and rSBP is a nonpolar cavity containing a proton acceptor that participates in a specific interaction, possibly a hydrogen bond, with the 3'-hydroxyl group of the bound steroid.     

Specific labeling of protein domains with antibody fragments

Monovalent antibody Fab fragments, prepared from antisera raised against two different types of crystalline arrays made of either intact, or a proteolytic fragment of bacteriophage T4 major capsid protein, gp23*, were employed to stoichiometrically label different gp23* protein domains on the outer surface of a tubular variant (i.e., "polyheads") of bacteriophage T4 capsids. Computer filtrations of both negatively stained and freeze-dried/metal-shadowed specimens permitted approximate mapping of the Fab binding sites within the capsomere of the polyheads.     

Observation and quantitation of metal-binding sites in the sex steroid-binding protein of human and rabbit sera using the luminescent probe terbium

The first direct evidence for specific metal-binding sites in pure human and pure rabbit sex steroid-binding protein (SBP) is obtained using the luminescent lanthanide terbium. Terbium, a probe for calcium sites in proteins, provided protection of the SBP steroid-binding activity in diluted human serum samples equivalent to that provided by calcium. Pure SBP, first treated with ethylenediaminetetraacetate, was dialyzed against buffer containing TbCl₃. After gel filtration to remove nonspecifically bound terbium, the protein was denatured in urea. The amount of protein-bound terbium was determined by luminescence enhancement of the lanthanide using the chelator dipicolinate, yielding four metal-binding sites per mole of dimer protein from both species.