O-Glycosylation of human sex hormone-binding globulin is essential for inhibition of estradiol-induced MCF-7 breast cancer cell proliferation.

Human sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein, and each SHBG monomer may have an O-linked oligosaccharide at Thr(7) and up to two N-linked oligosaccharides at Asn(351) and Asn(367). In addition, a common genetic variant of SHBG exists with an extra site for N-glycosylation at residue 327. In the present study, we isolated MCF-7 derived cell lines expressing human SHBG cDNAs encoding the wild type protein or various glycosylation mutants. Estradiol (1 nM) treatment of parental (untransfected) MCF-7 cells or MCF-7 cells transfected with control expression vectors resulted in an increase in proliferation which was fully abrogated by co-incubation with an equimolar amount of human SHBG. In contrast, the same amount of purified SHBG added to MCF-7 cells expressing wild type SHBG partially inhibited the estradiol-induced cell proliferation. A high affinity binding site for SHBG was detectable on untransfected and control cells, but not on MCF-7 cells expressing wild type SHBG. Moreover, the treatment of MCF-7 cells with the conditioned medium containing wild type SHBG caused the disappearance of the SHBG plasma membrane-binding site. Media containing SHBG N-glycosylation mutants exerted the same effect, but mutants lacking the O-linked oligosaccharide at Thr(7) failed to do so. Estradiol-induced proliferation of parental MCF-7 cells was also inhibited by treatment with conditioned medium containing wild type SHBG or SHBG mutants lacking N-linked oligosaccharides, or containing an additional N-linked oligosaccharide at residue 327. However, MCF-7 conditioned medium containing SHBG mutants lacking an O-linked oligosaccharide at Thr(7) failed to exert this effect. These data suggest that O-glycosylation of SHBG is essential for SHBG binding to a membrane receptor that is responsible for inhibiting the estradiol-induced proliferation of MCF-7 breast cancer cells.

Resolution of the human sex hormone-binding globulin dimer interface and evidence for two steroid-binding sites per homodimer.

Human sex hormone-binding globulin (SHBG) transports sex steroids in the blood. It functions as a homodimer, but there is little information about the topography of its dimerization domain, and its steroid binding stoichiometry is controversial. The prevailing assumption is that each homodimeric SHBG molecule contains a single steroid-binding site at the dimer interface. However, crystallographic analysis of the amino-terminal laminin G-like domain of human SHBG has shown that the dimerization and steroid-binding sites are distinct and that both monomers within a homodimeric complex are capable of binding steroid. To validate our crystallographic model of the SHBG homodimer, we have used site-directed mutagenesis to create SHBG variants in which single amino acid substitutions (V89E and L122E) were introduced to produce steric clashes at critical positions within the proposed dimerization domain. The resulting dimerization-deficient SHBG variants contain a steroid-binding site with an affinity and specificity indistinguishable from wild-type SHBG. Moreover, when equalized in terms of their monomeric subunit content, dimerization-deficient and wild-type SHBGs have essentially identical steroid binding capacities. These data indicate that both subunits of the SHBG homodimer bind steroid and that measurements of the molar concentration of SHBG homodimer in serum samples have been overestimated by 2-fold.

Steroid-binding specificity of human sex hormone-binding globulin is influenced by occupancy of a zinc-binding site.

One calcium-binding site (site I) and a second poorly defined metal-binding site (site II) have been observed previously within the amino-terminal laminin G-like domain (G domain) of human sex hormone-binding globulin (SHBG). By soaking crystals of this structure in 2.5 mm ZnCl(2), site II and a new metal-binding site (site III) were found to bind Zn(2+). Site II is located close to the steroid-binding site, and Zn(2+) is coordinated by the side chains of His(83) and His(136) and the carboxylate group of Asp(65). In this site, Zn(2+) prevents Asp(65) from interacting with the steroid 17beta-hydroxy group and alters the conformations of His(83) and His(136), as well as a disordered region over the steroid-binding site. Site III is formed by the side chains of His(101) and the carboxylate group of Asp(117), and the distance between them (2.7 A) is increased to 3.7 A in the presence of Zn(2+). The affinity of SHBG for estradiol is reduced in the presence of 0. 1-1 mm Zn(2+), whereas its affinity for androgens is unchanged, and chemically-related metal ions (Cd(2+) and Hg(2+)) have similar but less pronounced effects. This is not observed when Zn(2+) coordination at site II is modified by substituting Gln for His(136). An alteration in the steroid-binding specificity of human SHBG by Zn(2+) occupancy of site II may be relevant in male reproductive tissues where zinc concentrations are very high.

Crystal structure of human sex hormone-binding globulin: steroid transport by a laminin G-like domain.

Human sex hormone-binding globulin (SHBG) transports sex steroids in blood and regulates their access to target tissues. In biological fluids, SHBG exists as a homodimer and each monomer comprises two laminin G-like domains (G domains). The crystal structure of the N-terminal G domain of SHBG in complex with 5alpha-dihydrotestosterone at 1.55 A resolution reveals both the architecture of the steroid-binding site and the quaternary structure of the dimer. We also show that G domains have jellyroll topology and are structurally related to pentraxin. In each SHBG monomer, the steroid intercalates into a hydrophobic pocket within the beta-sheet sandwich. The steroid and a 20 A distant calcium ion are not located at the dimer interface. Instead, two separate steroid-binding pockets and calcium-binding sites exist per dimer. The structure displays intriguing disorder for loop segment Pro130-Arg135. In all other jellyroll proteins, this loop is well ordered. If modelled accordingly, it covers the steroid-binding site and could thereby regulate access of ligands to the binding pocket.

Novel human corticosteroid-binding globulin variant with low cortisol-binding affinity.

Corticosteroid-binding globulin (CBG) is the plasma transport protein that regulates the access of glucocorticoid hormones to target cells. Genetic deficiencies of CBG are rare, and only a single human CBG variant (Trancortin Leuven) has been related so far to decreased cortisol-binding affinity. We report here on a 43-yr-old woman, referred for chronic asthenia and hypotension, with repeatedly low morning serum cortisol levels (22-61 nmol/L; normal range, 204-546 nmol/L), normal plasma ACTH levels (38-49 pg/mL; normal, <50 pg/mL), and normal urinary cortisol (10-76 nmol/24 h; normal range, 10-105 nmol/24 h). An increased percent-free (dialysable fraction) serum cortisol (8.7-9.7%, normal range, 2.9-3.9%) suggested abnormal CBG binding activity. Indeed, she had a low serum CBG concentration (24 mg/L vs. 44+/-6 mg/L in normal women), and the affinity of her CBG for cortisol was decreased (association constant, Ka = 0.12 L/nmol vs. 0.82+/-0.29 L/nmol). In her immediate family members, the serum CBG concentration and cortisol-binding activity were normal in her husband, but the four living children had slightly lower serum CBG concentrations than the reference ranges for their pre- and postpubertal status. Measurements of cortisol distribution in undiluted serum indicated that an increase in the percentage of nonprotein-bound cortisol offsets the low cortisol levels to give approximately normal concentrations of free cortisol in serum. Direct sequencing of PCR-amplified exons encoding CBG revealed that the proband was homozygous for a polymorphism (GAC-AAC) in the codon for residue 367, which results in a Asp367-->Asn substitution. Her children were heterozygous for this polymorphism. When this nucleotide change was introduced into a normal human CBG complementary DNA, for expression in Chinese hamster ovary cells, Scatchard analysis demonstrated that the Asn367 substitution reduced the affinity of human CBG for cortisol by approximately 4-fold (Ka = 0.15 L/nmol), as compared to normal recombinant CBG (Ka = 0.66 L/nmol). These results suggest that Asp367 is an important determinant of CBG steroid-binding activity and that normal negative regulation of the hypothalamic-pituitary-adrenal axis is maintained by relatively normal serum-free cortisol concentrations, despite a marked reduction in the steroid-binding affinity of this novel human CBG variant, which we have designated as CBG-Lyon.

N-glycans are not the signal for apical sorting of corticosteroid binding globulin in MDCK cells.

It has been suggested that N-glycans act as a general sorting signal for secretory proteins in MDCK cells [Scheiffele et al. (1995) Nature 378, 96-98]. Human corticosteroid binding globulin contains six consensus sites for N-glycosylation and is known to be secreted to the apical side of MDCK cells. Our results show that wild-type corticosteroid binding globulin is N-glycosylated when it is recombinantly expressed in MDCK cells. Six mutants, each lacking one of the N-glycosylation sites, and a mutant lacking all six N-glycosylation sites were also secreted to the apical side of MDCK cells in a polarized manner. Thus, the N-glycans on corticosteroid binding globulin do not act as an apical sorting signal in MDCK cells.

Crystallization of the N-terminal domain of human sex hormone-binding globulin, the major sex steroid carrier in blood.

The amino-teminal laminin G-like domain of human sex hormone-binding globulin (SHBG), which contains the steroid-binding site and the dimerization domain, has been produced in Escherichia coli, purified to homogeneity and crystallized in complex with 5alpha--dihydrotestosterone (DHT) in two different crystal forms. Native data sets have been collected for tetragonal crystals (space group P4(1)22 or P4(3)22; unit-cell parameters a = 52.2, c = 148.4 A) diffracting to 3.3 A and trigonal crystals (R32; a = 104.0, c = 84.4 A) diffracting to better than 1.6 A. Since both crystal forms can only accommodate a single monomer in the asymmetric unit and share twofold rotational symmetry, it is proposed that the homodimer of this truncated form of SHBG, as observed in ultracentrifugation experiments, displays C(2) point-group symmetry.

Structure and function of corticosteroid-binding globulin: role of carbohydrates.

To study the site-specificity of human corticosteroid-binding globulin (CBG) glycosylation and the functional significance of individual carbohydrate chains in its molecule, a panel of recombinant CBG mutants containing each of the six potential glycosylation sites alone and in various combinations has been expressed in Chinese hamster ovary (CHO) cells. Analyses of these mutant glycoproteins showed that three of the glycosylation sites are only partially utilized, and this may contribute to the production of glycoforms with distinct physiological functions. Processing of individual carbohydrate chains (branching and fucosylation) is site-specific and may, thus, account for the formation of structural determinants essential for the recognition of CBG by cell membranes. Glycosylation at the only phylogenetically conserved consensus site, Asn238-Gly239-Thr240, is essential for the biosynthesis of CBG with steroid-binding activity. Evidence has been obtained to support the hypothesis that transient carbohydrate-polypeptide interactions between Trp266 and the maturing carbohydrate chain at Asn238 occur during early stages of the CBG biosynthesis which affect protein folding and formation of the steroid-binding site. Another tryptophan residue, Trp371, has been found to be critical for CBG-steroid interactions and is likely located in the steroid-binding site.

Substitutions of tryptophan residues in human corticosteroid-binding globulin: impact on steroid binding and glycosylation.

Human corticosteroid-binding globulin (CBG) contains four tryptophan residues at positions 141, 185, 266 and 371; one of which is thought to be located in the steroid-binding site. These residues were substituted by site-directed mutagenesis and expression of mutant CBG cDNAs in Chinese hamster ovary cells. Analyses of the resulting mutants indicate that Trp371 is most likely located in the steroid-binding site, and that hydrophobic interactions between Trp141 and the steroid molecule or other amino-acids in the CBG polypeptide may also contribute to high-affinity interactions between CBG and its steroid ligands. In addition, substitution of Trp266 resulted in altered glycosylation of CBG, and this supports the concept that it participates in intra-molecular carbohydrate-polypeptide interactions which may influence the conformation and secretion of this glycoprotein.

Glycosylation of human corticosteroid-binding globulin. Differential processing and significance of carbohydrate chains at individual sites.

Human corticosteroid-binding globulin (CBG) comprises 383 amino acids and six consensus sites for attachment of N-acetyllactosamine-type oligosaccharides. To study the extent of addition and processing of individual carbohydrate chains, we expressed CBG mutants, each containing only one of the six possible glycosylation sites, in Chinese hamster ovary cells and examined their electrophoretic, immunochemical, and lectin-binding properties. This indicated that Asn9, Asn308, and Asn347 are partially glycosylated and that oligosaccharides attached to Asn9, Asn238, Asn308, and Asn347 are predominantly biantennary, while more branched (most likely, triantennary) oligosaccharides are preferentially linked to Asn74 and Asn154. Only one of the biantennary chains (attached to Asn9) contains significant amounts of fucose. These data indicate that oligosaccharide processing is site-specific, and analyses of three other mutants, in which an additional glycosylation site was preserved, demonstrated that the processing of individual oligosaccharides occurs independently. Thus, the glycosylation of recombinant CBG appears to resemble that of natural human CBG. As we have previously found, glycosylation at Asn238 is essential for the production of CBG with steroid-binding activity, but when the mutant containing only one oligosaccharide at this position was enzymatically deglycosylated, its steroid-binding activity was unaltered. This suggests that interaction between this carbohydrate chain and the polypeptide is necessary for the folding and creation of the steroid-binding site only during CBG biosynthesis.

Glycosylation of human corticosteroid-binding globulin at aspargine 238 is necessary for steroid binding.

Human corticosteroid binding-globulin (CBG) is a plasma glycoprotein that binds and regulates the biological activity of glucocorticoids and progesterone. Carbohydrates comprise approximately 25% of its molecular mass being represented by bi- and triantennary N-linked oligosaccharides of the N-acetyllactosamine type. To assess the impact of these carbohydrate chains on CBG production and steroid binding, we mutated a human CBG cDNA so that the six consensus sites for N-glycosylation in the CBG polypeptide were eliminated individually and in various combinations. Expression of the mutant cDNAs in Chinese hamster ovary cells showed that all consensus sites may be utilized during the CBG biosynthesis and that the immunochemical properties of the recombinant glycoproteins are similar to those of CBG isolated from human serum. Removal of sugar chains generally led to a reduction in the secretion of recombinant CBG, but complete removal of N-glycosylation sites did not prevent production or secretion of the protein. Our data indicate that an oligosaccharide linked to Asn238 is essential for steroid binding, and we suggest that an interaction between this sugar chain and the polypeptide may be essential for the creation of a high affinity steroid-binding site. In addition, concanavalin A chromatography of mutants containing only one N-glycosylation site at either Asn74 or Asn238 indicated that processing of the oligosaccharides at these positions is site-specific.

A transcortin-binding protein in the plasma membrane of human syncytiotrophoblast.

Using affinity chromatography on immobilized transcortin of 125I-labeled, cholate-solubilized plasma membranes of human syncytiotrophoblast, a transcortin-binding protein with a minimal Mr of about 20 kDa has been isolated. It was found to be a sialoglycoprotein with an isoelectric point at pH 4.4 (about 5.0 after the treatment with neuraminidase). We assume that this protein is a component of membrane recognition system for transcortin-steroid complexes.

On the functional form of transcortin-recognizing subunit of transcortin membrane receptor.

Complex formation between transcortin and the 20 kDa sialoglycoprotein from the plasma membrane of human decidual endometrium (presumably a transcortin-recognizing subunit of transcortin membrane receptor) was studied using cross-linking reagents. The action of 1,5-difluoro-2,4-dinitrobenzene (DFDNB) on a solution of 125I-labelled 20 kDa sialoglycoprotein and unlabelled transcortin resulted in the formation of two 125I-containing containing species that corresponded to covalently linked complexes of one transcortin molecule and either 2 or 4 molecules of the labeled membrane sialoglycoprotein. Only the latter complex was observed when the endometrium membranes were incubated with [125I]transcortin and treated with DFDNB. This suggests that the functional form of transcortin-recognizing subunit of the membrane receptor is a tetramer.

Interaction of human CBG with cell membranes.

Specific binding sites for corticosteroid-binding globulin (CBG) and its pregnancy-associated variant (pCBG), having a modified carbohydrate moiety, were found in the plasma membranes of human liver, decidual endometrium and placental syncytiotrophoblast. The membrane binding was influenced by the conformation of the glycoprotein molecules and structure of their carbohydrate chains. CBG receptor was solubilized from the endometrium membrane and partially characterized. It was found to have a subunit structure, with a homooligomeric sialoglycoprotein consisting of four 20 kDa protomeric species being involved in the recognition of the CBG molecules complexed with progesterone or cortisol. A kinetic study using membrane microvesicles derived from the syncytiotrophoblast brush border revealed that neither CBG nor pCBG restricted cortisol accumulation in the intravesicular space, whereas only normal CBG could penetrate the syncytiotrophoblast membrane. Action of the CBG-cortisol complex on trophoblast cells resulted in the activation of membrane adenylate cyclase and growth of the cAMP accumulation within these cells. Collectively, these findings suggest that both normal CBG and pCBG are involved in the guided transport of steroid hormones to the target cells and transmembrane transfer of hormones and/or hormonal signals.

Transcortin does not restrict the transmembrane transfer of cortisol.

We have studied the specific binding of both free and transcortin-bound cortisol to the microvesicles derived from the brush border of the plasma membrane of human placental syncytiotrophoblast. Kinetics of the steroid binding to these microvesicles was found to be independent on cortisol being complexed with transcortin. Both cortisol and transcortin were accumulated in the inner space of the microvesicles. This suggests that transcortin-cortisol complex penetrates the plasma membrane and the transcortin-bound steroid can thus enter syncytiotrophoblast and exert its hormonal effects on this tissue.

Binding of human sex hormone-binding globulin-androgen complexes to the placental syncytiotrophoblast membrane.

We have found that human SHBG complexed with androgens binds specifically to the plasma membrane of human placental syncytiotrophoblast. Apparent equilibrium association constants were 5.3.10(11) M-1 for SHBG-testosterone complex and 1.1.10(11) M-1 for SHBG-5 alpha-dihydrotestosterone. Devoid of steroid, SHBG did not bind to the membrane. This suggests that the specific membrane binding of SHBG-androgen complexes is a step of the mechanism of androgen action on syncytiotrophoblast.

Study of the transcortin binding to human endometrium plasma membrane.

Transcortin complexed with progesterone was shown to bind specifically to the plasma membrane of human decidual endometrium. The binding reaction was characterized by a high affinity (an apparent Kd value was (1.0 +/- 0.2).10(-10) mol/l) and high selectivity: such human serum proteins as albumin, orosomucoid, transferrin, thyroxine-binding globulin and sex hormone-binding globulin did not compete with transcortin for the membrane binding sites. Transcortin binding to the membrane was steroid-dependent: transcortin-cortisol complex bound to the membranes substantially more weakly than transcortin-progesterone, and specific binding of transcortin devoid of steroid was not detected. Using a radioimmunoassay, we have measured the concentration of endogenous transcortin in highly purified membrane preparations solubilized with sodium cholate. It was found that an extensive washing of decidual strips with a physiological buffer prior to the membrane isolation resulted in a decrease of the endogenous transcortin level along with an increase of the specific membrane binding of exogenous 125I-labeled transcortin. Affinity chromatography on immobilized transcortin was used to isolate transcortin-binding components from 125I-labeled, cholate-solubilized plasma membrane of decidual endometrium. Along with lipid components, the structure of which was not investigated, a 125I-labeled transcortin-binding sialoglycoprotein with a minimal Mr of 20.0 +/- 1.5 kDa and a pI of approx. 3.3 was detected. In the presence of transcortin, this sialoglycoprotein could be precipitated with a monospecific antitranscortin antiserum. Using hydroxylapatite as a separating agent, the interaction of transcortin and the membrane sialoglycoprotein in model systems containing the two proteins and various steroid hormones was studied. It was found that the membrane sialoglycoprotein displayed a higher affinity for transcortin-progesterone than for transcortin-cortisol (the Kd values were, respectively, 2.10(-11) and 7.10(-11) mol/l) and it did not bind transcortin complexed with testosterone.

[Characteristics of a transcortin-binding component of the plasma membrane of cells of the human decidual endometrium]. / Kharakteristika transkortin-sviazyvaiushchego komponenta plazmaticheskoi membrany kletok detsidual'nogo éndometriia cheloveka.

A sialoglycoprotein was isolated by affinity chromatography on immobilized transcortin from plasma membranes of human decidual endometrium cells, whose components were labeled with 125I and solubilized with sodium cholate. The apparent molecular mass of the monomer is 20.0 +/- 1.5 kDa, pI is at pH 3.3. The sialoglycoprotein specifically binds transcortin complexed to progesterone with Kd approximately 10(-10) M.

[Variety of transcortin in the blood of women during normal pregnancy]. / Raznovidnost' transkortina v krovi zhenshchin pri normal'noi beremennosti.

A concentration of pregnancy-related transcortin variety was detected in the venous blood serum of women at varying time of normal pregnancy and after delivery as well as in umbilical and retroplacental blood serum using a radio-immunoassay. This transcortin variety was found in the blood at early stages of pregnancy (the end of the 1st-the start of the 2nd trimester). During pregnancy the content of this variety increased, mainly in the 2nd trimester. At the end of pregnancy transcortin variety concentration achieved values typical of retroplacental blood serum, corresponding to approximately 10% of total transcortin concentration. The absence of transcortin variety in the umbilical blood serum and a slight decrease in its level in the mother's blood by the 5th day after delivery can be suggestive of the fact that this protein is synthesized in the mother's body and does not penetrate the placental barrier.