Properties and serum levels of pregnancy-associated variant of human transcortin.

The amino acid composition, N- and C-terminal amino acid sequences, and the basic physicochemical and immunochemical properties of the recently discovered pregnancy-associated molecular variant of human transcortin (Strel'chyonok, O.A., Avvakumov, G.V. and Akhrem, A.A. (1984) Carbohydr. Res. 134, 133-140) have been found to be identical to those of transcortin from normal donor serum. This suggests the identity of polypeptide moieties of the two glycoproteins. The transcortin variant has a lower isoelectric point (3.5-4.1) than normal transcortin (3.6-4.2), and different electrophoretic mobility in low-porosity polyacrylamide gel (one band versus two for normal transcortin). These differences can be reasonably explained by different organization of the carbohydrate moieties of these glycoproteins due to diverse post-translational modification of a single polypeptide chain. The levels of transcortin variant in the maternal venous serum throughout normal gestation (447 donors in all) and on the fifth day after delivery, as well as in umbilical cord serum and extracts of term placenta, have been measured by a radioimmune assay. Analysis of the data obtained allowed us to conclude that the biosynthesis of pregnancy-associated transcortin variant occurs in some organ of the maternal organism rather than in the feto-placental system, and it is a characteristic of pregnancy as a unique physiological state of the female organism rather than a phenomenon caused by individual features of certain women. We assume that the transcortin variant takes part in the guided transport of corticosteroids and/or progestins into some tissues that develop in the course of gestation.

Evidence for the involvement of the transcortin carbohydrate moiety in the glycoprotein interaction with the plasma membrane of human placental syncytiotrophoblast.

We have studied the interaction of human transcortin and the pregnancy-associated transcortin variant with the microvesicular membrane fraction derived from the human placental syncytiotrophoblast. Two classes of specific binding sites for these glycoproteins were found in this membrane preparation. One of these displays a relatively high binding capacity, Bmax = 140 +/- 60 fmol transcortin per mg membrane protein, and a significantly higher affinity for transcortin, Kd = (1.6 +/- 0.6).10(-10) mol/l, than for the pregnancy-associated variant, Kd = (4.5 +/- 1.2).10(-9) mol/l. On the contrary, another class of the binding sites, occurring in the membranes at a far lower concentration: Bmax = 3.0 +/- 2.2 fmol transcortin per mg membrane protein, shows a higher affinity for the pregnancy-associated transcortin variant, Kd = (3.3 +/- 2.0).10(-12) mol/l, than for normal transcortin, Kd = (2.5 +/- 0.7).10(-11) mol/l. Since the pregnancy-associated variant differs from normal transcortin with respect to its carbohydrate structures only (Avvakumov, G.V. and Strel'chyonok, O.A. (1987) Biochim. Biophys. Acta 925, 11-16), the results of the present work suggest that the transcortin carbohydrates are directly involved in the specific interaction of this serum hormone-binding globulin with the plasma membrane of the placental syncytiotrophoblast.

Evidence for the presence of specific binding sites for transcortin in human liver plasma membranes.

Binding sites which recognize and bind specifically asialotranscortin and the native transcortin-cortisol complex have been found in plasma membranes of human liver cells. The native conformation of transcortin is an absolute requirement for the binding reaction of the transcortin-hormone complex. Sex-hormone-binding globulin and thyroxine-binding globulin from human serum do not bind to this binding sites.

Subcellular distribution and selectivity of the protein-binding component of the recognition system for sex-hormone-binding protein-estradiol complex in human decidual endometrium.

In order to assess the subcellular distribution of the protein-binding component of the recognition system for sex-hormone-binding protein-estradiol complex (Strel'chyonok, O.A., Avvakumov, G.V. and Survilo, L.I. (1984) Biochim. Biophys. Acta 802, 459-466), plasma membranes and other subcellular fractions were prepared from homogenate of human decidual endometrium (8-12 weeks of pregnancy). Specific binding of 125I-labeled sex-hormone-binding protein complexed with estradiol was found preferentially in the plasma membrane fraction. The protein-binding component of the recognition system was found to specifically bind sex-hormone-binding protein complexed with estrogens (estradiol, estriol, estrone, with close affinities) but not with androgens (testosterone and 5 alpha-dihydrotestosterone). Specific membrane binding of 125I-labeled sex-hormone-binding protein complexed with 17 alpha-pregna-2,4-dien-20-yn-[2,3-d]isoxazol-17-ol (danazol) was also detected. This allowed us to suggest a putative mechanism for the danazol pharmacological action on endometrium which involves the interaction of the steroid-protein complex with the plasma membrane of endometrial cells. It is proposed that sex-hormone-binding protein takes part in the guided transport of steroids into endometrial cells.

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.

A recognition system for sex-hormone-binding protein-estradiol complex in human decidual endometrium plasma membranes.

We have studied the specific binding of human serum sex-hormone-binding protein and its complexes with estradiol and testosterone to an enriched membrane fraction isolated from human decidual endometrium. The specific binding of sex-hormone-binding protein to these membranes has been found to be dependent on it being bound with estradiol. When estradiol concentration in the medium is high enough to provide for the saturation of sex-hormone-binding protein with the steroid, the protein exhibits specific binding to the membranes with an apparent equilibrium constant, Kd = (3.5 +/- 2.0) X 10(-12) M. In the absence of estradiol, the binding protein devoid of steroid or complexed with testosterone does not interact with the membranes. At low concentrations of sex-hormone-binding protein and estradiol, when their complex is almost completely dissociated in solution, it can be reconstituted on the membranes with the optimum estradiol to binding protein ratio being 1:1 (mol/mol). It is proposed that, in plasma membranes of the estradiol target cells, there is a recognition system for the sex-hormone-binding protein-estradiol complex which may allow these cells to take up from blood not only free estradiol, but also estradiol complexed with the binding protein.

Organization of the transcortin-binding domain on placental plasma membranes.

Complex formation between transcortin (corticosteroid-binding globulin) and 20 kDa sialoglycoprotein from human syncytiotrophoblast plasma membranes (presumably a transcortin-recognizing subunit of the transcortin membrane receptor) was studied using FPLC and cross-linking with bifunctional reagents. The action of 1,5-difluoro-2,4-dinitrobenzene (DFDNB) on a solution of the purified 20 kDa sialoglycoprotein and transcortin resulted in formation of covalently linked complexes of 95 kDa and 140 kDa consisting of one transcortin molecule and either two or four molecules of the membrane sialoglycoprotein (the molecular mass of transcortin is 55 kDa). Additionally, cross-linking resulted in the appearance of a 43 kDa species which is the cross-linked dimer of the membrane protein. The dimer was also observed during chromatography on a Superose 12 column in the absence of DFDNB treatment. Treatment of intact syncytiotrophoblast membranes with DFDNB resulted in isolation of the transcortin binding protein dimer as the major portion of total pool of the protein. Formation of the transcortin complexes with two and four molecules of the membrane protein was also observed when the membranes were incubated with 125I-labeled transcortin and treated with DFDNB, but formation of the latter complexes predominated. The results obtained suggest that the recognizing and binding domain for transcortin in placental membranes is organized as dimers consisting of non-covalently linked sialoglycoprotein monomers of a 20 kDa each and that transcortin has two sites for interaction with this dimer. Apparently, binding of two dimers results in the formation of the functional form of the transcortin-receptor complex. The possible biological role of such a complex is discussed.

Study of the carbohydrate moiety of human serum sex hormone-binding globulin.

Sex hormone-binding globulin from human blood serum contains two biantennary N-linked oligosaccharide chains of the N-acetyllactosamine type and one O-linked oligosaccharide per one molecule of the glycoprotein. These conclusions have been based on the results of methylation analysis of the whole glycoprotein and investigation of the structures of its glycopeptides prepared using pronase digestion.

Structural organization of the carbohydrate moiety of human transcortin as determined by methylation analysis of the whole glycoprotein.

Methylation analysis of human transcortin showed that this glycoprotein contains N-glycosidically linked oligosaccharide chains of N-acetyllactosamine type, most of the chains being biantennary and others tri- and/or tetra-antennary. The carbohydrate chains of transcortin are also heterogeneous with respect to the content of fucose and the position of the glycosidic linkages.

Isolation and characterization of glycopeptides of human transcortin.

Pronase digestion of human transcortin yielded two major glycopeptides (GID3 and G2D2, in a molar ratio of approx. 1.0 to 1.5) which were isolated by gel and anion-exchange chromatography. Chromatography behaviour, methylation analysis and analytical chromatography on a Con A-Sepharose column suggested that both of the glycopeptides were N-linked asparaginyl oligosaccharides of the N-acetyllactosamine type, G1D3 and G2D2 being triantennary and biantennary isoglycans, respectively. Microheterogeneity in regard to the position of the linkages between sialyl and galactosyl residues occurred in the triantennary and, possibly, in biantennary isoglycans.

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.

Methylation analysis in glycoprotein chemistry: a comparative study of the carbohydrate structures of three hormone-binding glycoproteins from human serum.

The structures of the carbohydrate moieties of three hormone-binding glycoproteins from human serum, namely, thyroxine-binding globulin, transcortin, and sex hormone-binding globulin, have been characterised using quantitative g.l.c. of the methylated monosaccharide derivatives obtained after methanolysis of the methylated glycoproteins.

Pregnancy-associated molecular variants of human serum transcortin and thyroxine-binding globulin.

Affinity chromatography on immobilised concanavalin A revealed that transcortin and thyroxine-binding globulin isolated from human postpartum serum contained approximately 10% of molecular variants that did not occur in these glycoproteins isolated from normal donor serum (both male and female). The chromatographic behaviour of the pregnancy-associated glycoprotein variants, their monosaccharide compositions, and the results of methylation analysis indicated that these variants contained only triantennary oligosaccharide chains of the N-acetyl-lactosamine type.

Testosterone destroys the transcortin-receptor complex.

Dissociation of the complex of transcortin receptor with immobilized transcortin in the presence of 10(-5) M testosterone has been shown with the use of affinity chromatography on transcortin-Sepharose. The specificity of this effect is confirmed by its abrogation in the presence of cortisol. The testosterone effect has been used for the elution of transcortin receptor from affinity column. The receptor retained transcortin-binding capacity after the elution and removal of testosterone. Characteristics of the receptor obtained by testosterone elution were identical with those of the transcortin eluted preparation.

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.

Methylation analysis in glycoprotein chemistry. General procedure for quantification of the products of solvolysis of permethylated glycopeptides and glycoproteins.

A technique for the gas chromatographic analysis of the products of solvolysis of permethylated glycopeptides and glycoproteins has been developed. It involves methanolysis of a permethylated compound, quantitative transformation of methyl ethers of methyl glycosides into the corresponding O-trimethylsilyl(TMS)-O-methylalditols [2-deoxy-2-(N-methyl)acetamido-O-TMS-O-methylalditols in the case of hexosamine derivatives] and gas chromatographic quantification using a single column packed with 0.4% OV-225 on surface-modified Chromosorb.

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.

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.