Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.     

Search for peptide immunogens of the beta-subunit of human chorionic gonadotropin (hCG) capable of eliciting hormone specific and neutralizing antisera. Identification of an undecapeptide eliciting hCG-specific antisera.

The work reported herein describes our attempts to identify peptide immunogens of the beta-subunit of the pregnancy-specific placental hormone, human chorionic gonadotropin (hCG), capable of eliciting hormone specific and neutralizing antisera. Hydrophilicity profiles of the beta-subunits of hCG and the homologous pituitary hormone, human luteinizing hormone (hLH) were compared and two sequences which are hydrophilic but unique to hCG-beta were identified. They are 6-12 and 109-119 of hCG-beta. Results of the studies with the undecapeptide 109-119 of hCG-beta are reported in this paper. The undecapeptide amide was synthesized using the p-(acyloxy) benzhydrylamine resin and antisera to the peptide were elicited in rabbits using the peptide-diphtheria toxoid conjugate. The peptide is highly immunogenic as both the rabbits responded with high titers of antibodies to the peptide. The antipeptide antibodies bound to hCG but not to hLH showing thereby that the region 109-119 of hCG-beta is a unique determinant of hCG. However, the antibodies were found not to neutralize the biological activity of hCG.     

Upregulation of placental indoleamine 2,3-dioxygenase by human chorionic gonadotropin

We tested the hypothesis that hCG can upregulate human trophoblast indoleamine 2, 3-dioxygenase (INDO), which catalyzes the breakdown of tryptophan in villous circulation. The results revealed that it can. Treatment of human trophoblasts with hCG resulted in a time and dose dependent increase in INDO mRNA and protein levels and its enzyme activity. The hCG effect was hormone specific and required the dimer conformation of hCG. The hCG effect required its receptors and was mediated by a cAMP dependent, but protein kinase A independent, mitogen-activated protein kinase 3/1 (MAPK3/1) signaling mechanism. In summary, the present data demonstrate a novel hCG effect on human placental INDO, which probably plays a key role at maternal fetal interface in preventing fetal rejection.     

Cytological distribution of chorionic gonadotropin subunit and placental lactogen messenger RNA in neoplasms derived from human placenta

Normal trophoblast of the human placenta elaborates at least two major protein hormones, chorionic gonadotropin (hCG), and placental lactogen (hPL). There are several gestational trophoblastic diseases of the placenta called hydatidiform mole, invasive mole, and choriocarcinoma. Molar and choriocarcinoma tissues characteristically synthesize large amounts of hCG and small quantities of hPL. To examine the role of trophoblast differentiation in the expression of the hCG and hPL genes, we studied the cytological distribution of their messenger RNA (mRNA) in tissue sections of human hydatidiform mole and choriocarcinoma by in situ hybridization. Histologically, these tissues are in different stages of cellular differentiation. In normal placenta, hCG alpha and - beta mRNA can be localized to some cytotrophoblasts and primarily to the syncytium, whereas hPL mRNA appears only in the syncytial layer. In hydatidiform mole, which still retains placental villous morphology, the hPL gene and hCG alpha and -beta genes are expressed but are poorly localized because of the admixture of cyto- and syncytiotrophoblasts. By contrast, choriocarcinoma, which is devoid of placental villous pattern but in which the cyto- and syncytiotrophoblast-like components are distinguishable, expresses hCG alpha and -beta in the syncytial- like areas but little, if any, hPL. These results suggest that a certain level of trophoblast differentiation, such as villous formation, is associated with hPL expression, while the hCG alpha gene and the hCG beta gene can be expressed in more disorganized tissues that contain cytotrophoblastic elements.     

Steroid-protein interaction in human placenta

Human placenta produces a large variety of bioactive substances with endocrine and neural competence: pituitary and gonadal hormones, hypothalamic-like releasing or inhibiting hormones, growth factors, cytokines and neuropeptides. The most recent findings indicate that locally produced hormones regulate the secretion of other placental hormones supporting a paracrine/autocrine regulation. In placental endocrinology, a particular relevance is played by steroid hormones. In fact, a specific gonadotropin-releasing hormone (GnRH)-human chorionic gonadotropin (hCG) regulation of placental steroidogenesis has been proposed as a placental internal regulatory system acting on steroids production from human placenta. In addition, activin and inhibin have been proposed as further regulatory substances of the synthesis and secretion of steroids; the addition of activin A to placental culture augments GnRH, hCG and progesterone, and this effect can be significantly reduced by the addition of inhibins. Finally, a steroid-steroid interaction is suggested by the evidence that placental estrogen has a positive role in the regulation of progesterone biosynthesis. Other steroid-protein interactions have been observed in human placenta. In fact, recent data indicate that progesterone inhibits placental corticotropin-releasing factor (CRF) and estrogens act on placental conversion of cortisol to cortisone, activating cortisol secretion by the fetal adrenal and enhancing fetal adrenal function with advancing gestation.     

Calcitriol affects hCG gene transcription in cultured human syncytiotrophoblasts

Background  

In pregnancy, maternal serum concentrations of calcitriol significantly rise as a result of increased renal and placental contribution in order to assure calcium supply for the developing fetus. Considering that placenta is a site for vitamin D activation, and the versatility and potency of calcitriol, it is feasible that this hormone participates in fetal/placental development and physiology. In the present work we studied calcitriol actions upon human chorionic gonadotropin (hCG) secretion and expression in cultured trophoblasts, as well as vitamin D receptor (VDR) and CYP27B1 immunolocalization in placental villi.     

Molecular cloning and characterization of a novel angiopoietin family protein, angiopoietin-3

Using homology-based PCR, we have isolated cDNA encoding a novel member (491 amino acids) of the angiopoietin (Ang) family from human adult heart cDNA and have designated it angiopoietin-3 (Ang3). The NH2-terminal and COOH-terminal portions of Ang-3 contain the characteristic coiled-coil domain and fibrinogen-like domain that are conserved in other known Angs. Ang3 has a highly hydrophobic region at the N-terminus (approximately 21 amino acids) that is typical of a signal sequence for protein secretion. Ang3 mRNA is most abundant in adrenal gland, placenta, thyroid gland, heart and small intestine in human adult tissues. Additionally, Ang3 is a secretory protein, but is not a mitogen in endothelial cells.     

Differentiation in human amniotic fluid cell cultures: chorionic gonadotropin production.

Two of the distinguishable cell classes subcultured from human amniotic fluid were examined for their capability to produce human chorionic gonadotropin (hCG) as determined by radioimmunoassay. The class that predominates in most cultures used for prenatal genetic diagnosis, previously termed AF (for amniotic fluid), secretes hCG into the culture medium. Dermal fibroblasts do not, nor does another type of cultured cell from amniotic fluid, previously termed F because of a resemblance to fibroblasts. Primary AF cultures produce more hCG than do subcultures. Evidence that this hormone is intact hCG is provided by its immunoreactivity with antisera raised against the beta-subunit and against the intact molecule of hCG. Furthermore, a dose-response curve for hormone in culture medium is parallel to that of highly purified intact hCG. It is postulated that AF cultures are derived from fetal membranes and retain properties of trophoblast.     

Heterogeneity of free alpha-subunit in term placenta

Free alpha-subunit in normal term placenta was examined for molecular weight, electric charge and ability to combine with standard hCG-beta in comparison with extracellular free alpha-subunit and standard hCG-alpha dissociated from urinary hCG in vitro. The gel chromatography on Sephadex G-100 of the placental extract revealed three major immunoreactive hCG-alpha peaks, designated as P alpha-A (Kav = 0.32-0.46), P alpha-B (0.47-0.58) and P alpha-C (0.59-0.70), near the position of standard hCG-alpha. In the isoelectric focusing, while P alpha-A was mainly distributed over the acidic region, the major components of P alpha-B and P alpha-C were distributed over the basic region. Furthermore, in the combination study with standard hCG-beta, such a alpha-subunit with acidic pI scarcely showed any combining activity whereas alpha-subunit with basic pI revealed significant combining activity. These results suggest the following possibilities: that 1) the various size species of placental alpha-subunit may be responsible for the progressive glycosylation; 2) the small alpha-subunit with basic pI may combine with beta-subunit to form immunoreactive hCG; 3) the alpha-subunit, which has not associated with beta-subunit, may be converted to a large and incombinative form with acidic pI by further glycosylation, followed by secretion as a free alpha-subunit.     

Expression of adrenocorticotropin-releasing hormone precursor gene in placenta and other nonhypothalamic tissues in man

Adrenocorticotropin-releasing hormone (CRH) is a peptide originally isolated from the hypothalamus. Immunocytochemical and RIA studies have revealed that CRH-like peptide is also localized in human nonhypothalamic tissues and some tumors. To see if CRH is synthesized in these nonhypothalamic tissues and tumors, we examined preproCRH mRNA in these tissues by Northern blot analysis using a cloned human preproCRH gene as a probe. PreproCRH mRNA was detected in human hypothalamus, cerebral cortex, adrenal gland, placenta, pheochromocytoma, and thymic carcinoid. The content of preproCRH mRNA in placenta was apparently greater than that in the whole hypothalamus.     

Human immunodeficiency virus type 1 infection of human placenta: potential route for fetal infection.

To determine the potential role of the placenta in transmission of human immunodeficiency virus (HIV) from mother to fetus, the ability of human placental tissue to support HIV type 1 (HIV-1) infection was examined. HIV-1-seronegative first-trimester placentas were maintained in culture and infected with HIV-1. Virus production, measured by HIV-1 antigen release into the supernatant, and HIV-1 DNA, identified by polymerase chain reaction, were detected for at least 12 days postinfection. Western immunoblot analysis showed Gag proteins, precursor p55, and cleavage products p24 and p17 in HIV-1-infected tissues. Double labeling of placental villi with antibodies to CD4 and placental trophoblast-specific alkaline phosphatase indicated that trophoblasts express CD4 antigen. Additionally, immunostaining of HIV-1-infected tissues with anti-p24 antibodies demonstrated HIV-1 protein expression in placental trophoblasts. Evaluation of human chorionic gonadotropin and progesterone production by the placental cultures indicated that there was a 90% decrease in human chorionic gonadotropin and a 70% decrease in progesterone production in HIV-1-infected cultures in comparison with controls. These data demonstrate that trophoblastic cells of human placenta tissue express CD4 and are susceptible to HIV-1 infection; also, placental endocrine function is decreased by HIV-1 infection. Thus, the placenta may serve as a reservoir of HIV-1 infection during pregnancy contributing to infection of the fetus, and decreased placental hormone production may result in impaired fetal development.     

SNAT4 isoform of system A amino acid transporter is expressed in human placenta

The system A amino acid transporter is encoded by three members of the Slc38 gene family, giving rise to three subtypes: Na+-coupled neutral amino acid transporter (SNAT)1, SNAT2, and SNAT4. SNAT2 is expressed ubiquitously in mammalian tissues; SNAT1 is predominantly expressed in heart, brain, and placenta; and SNAT4 is reported to be expressed solely by the liver. In the placenta, system A has an essential role in the supply of neutral amino acids needed for fetal growth. In the present study, we examined expression and localization of SNAT1, SNAT2, and SNAT4 in human placenta during gestation. Real-time quantitative PCR was used to examine steady-state levels of system A subtype mRNA in early (6-10 wk) and late (10-13 wk) first-trimester and full-term (38-40 wk) placentas. We detected mRNA for all three isoforms from early gestation onward. There were no differences in SNAT1 and SNAT2 mRNA expression with gestation. However, SNAT4 mRNA expression was significantly higher early in the first trimester compared with the full-term placenta (P < 0.01). We next investigated SNAT4 protein expression in human placenta. In contrast to the observation for gene expression, Western blot analysis revealed that SNAT4 protein expression was significantly higher at term compared with the first trimester (P < 0.05). Immunohistochemistry and Western blot analysis showed that SNAT4 is localized to the microvillous and basal plasma membranes of the syncytiotrophoblast, suggesting a role for this isoform of system A in amino acid transport across the placenta. This study therefore provides the first evidence of SNAT4 mRNA and protein expression in the human placenta, both at the first trimester and at full term.