Glycan characterization of pregnancy-specific glycoprotein 1 and its identification as a novel Galectin-1 ligand.

Pregnancy-specific beta 1 glycoprotein (PSG1) is secreted from trophoblast cells of the human placenta in increasing concentrations as pregnancy progresses, becoming one of the most abundant proteins in maternal serum in the third trimester. PSG1 has seven potential N-linked glycosylation sites across its four domains. We carried out glycomic and glycoproteomic studies to characterize the glycan composition of PSG1 purified from serum of pregnant women and identified the presence of complex N-glycans containing poly LacNAc epitopes with α2,3 sialyation at four sites. Using different techniques, we explored whether PSG1 can bind to galectin-1 (Gal-1) as these two proteins were previously shown to participate in processes required for a successful pregnancy. We confirmed that PSG1 binds to Gal-1 in a carbohydrate-dependent manner with an affinity of the interaction of 0.13 µM. In addition, we determined that out of the three N-glycosylation-carrying domains, only the N and A2 domains of recombinant PSG1 interact with Gal-1. Lastly, we observed that the interaction between PSG1 and Gal-1 protects this lectin from oxidative inactivation and that PSG1 competes the ability of Gal-1 to bind to some but not all of its glycoprotein ligands.

Aborto diferido como origen de una enfermedad trofoblástica persistente / Persistent trophoblastic disease presenting as missed abortion

La enfermedad trofoblástica persistente puede ser diagnosticada después de cualquier tipo de gestación, pero su frecuencia aumenta tras el hallazgo de un embarazo molar, con lo que su adecuado diagnóstico y seguimiento es vital para un tratamiento temprano y eficaz. Presentamos un caso de un aborto diferido, en el que se diagnosticó mola completa y tras la confirmación de persistencia de esta, precisó de tratamiento poliquimioterápico para lograr su curación (AU)

Persistent trophoblastic disease can be diagnosed after any type of pregnancy, but the frequency of this entity increases after molar pregnancies and consequently early diagnosis and follow-up is vital for prompt and effective treatment. We present a case of missed abortion, with diagnosis of a complete hydatiform mole. Persistent trophoblastic disease was subsequently diagnosed, requiring treatment with multiple drug therapy to achieve complete resolution (AU)

Purification and characterization of a bovine pregnancy-associated glycoprotein.

A 67000 Mr bovine pregnancy-associated glycoprotein (bPAG) has been isolated from fetal cotyledons and purified to homogeneity by HPLC. The purification was monitored by a double immunodiffusion test and by RIA in conjunction with an antiserum raised against a crude fraction of placenta-specific antigens. The molecular weight of bPAG was estimated to be 67000 by SDS-PAGE. The isoelectric points (pI) of the four isoforms, determined by high-resolution analytical electrofocusing in polyacrylamide gel, were 4.4, 4.6, 5.2, and 5.4. The carbohydrate content of the bPAG consisted of approximately 10.02 +/- 1.09% neutral sugar and variant amounts of sialic acid (from 0.29 +/- 0.06% in the most basic isoform to 2.1 +/- 0.31% in the most acidic isoform). A specific antiserum was raised against the purified bPAG. A specific RIA showed that the bPAG was antigenically unrelated to BSA, alphafetoprotein (AFP), and human schwangerschafts-spezifischen (pregnancy-specific) beta 1 glycoprotein (SP1). According to some characteristics (e.g. the molecular weight), the purified bPAG may correspond to a form of the pregnancy-specific protein B previously described by Sasser and colleagues (Biol Reprod 1986; 35:936-942).

Correlación anatomoclínica de la enfermedad trofoblastica 1988-1991 / Anatomoclinic correlation of trophoblastic diseasse

Se estudiaron 50 casos de enfermedad trofoblastica, analizando cada caso en forma individual, según edad, paridad, procedencia, sintomás, diagnóstico, resolución del cuadro control y clasificación. Con una frecuencia del 56 se presento en pacientes cuyas edades oscilan entre 15 y 25 años. En el 94 procedian del area sub urbana y rural, solo en el 6 eran del area urbana. De acuerdo a la edad gestacional el 86, correspondio a mas de 11 semanas de gestación. El sangrado genital y el dolor abdominal se presento en el 100 de las pacientes. En el 50 de los casos se resolvieron por inductoconducción y legrado. De acuerdo al diagnóstico histopatológico en el 72 correspondio a Mola Hidatiforme, en el 4 a Coriocarcinoma, el resto no tenia este examén.

Characterization of two new members of the pregnancy-specific beta 1-glycoprotein family from the myeloid cell line KG-1 and suggestion of two distinct classes of transcription unit.

Pregnancy-specific beta 1-glycoproteins (PSGs) represent a large group (approximately 12-15) of proteins, related to members of the carcinoembryonic antigen family, that are abundant in placental tissue and in the sera of pregnant women. We describe the isolation and characterization of two additional PSG cDNAs, PSG9 and PSG10, whose transcripts are largely expressed in placental tissue and to a lesser extent in some other cell types, including myeloid cells differentiated to granulocytes. PSG9 and PSG10 are representatives of two distinct classes of PSG protein that have N-termini with or without the Arg-Gly-Asp motif implicated in adhesion. In addition to this distinction at the amino acid level, our analysis of several PSG cDNAs suggests that the transcription units encoding these proteins may be further distinguished in their 3' untranslated sequences, thus suggesting possibilities for transcriptional regulation of the two major protein classes.

[Comparative physico-chemical characteristics of trophoblastic beta-1 glycoprotein preparations isolated from retroplacental blood]. / Sravnitel'naia fiziko-khimicheskaia kharakteristika preparatov trofoblasticheskogo beta1-glikoproteina, vydelennykh iz retroplatsentarnoi krovi.

Trophoblastic beta 1-glycoprotein preparations (TBG-1, TBG-2) isolated by different methods preserve their physicochemical and immunochemical properties. According to the data of disk-electrophoresis and densitometry TBG-1 preparation obtained using two-stage method is highly purified and protein yield is about 20%, while TBG-2 isolated by multi-stage method has a small number of admixture proteins.

Effects of sodium butyrate on the synthesis of human pregnancy-specific beta 1-glycoprotein.

Human pregnancy-specific beta 1-glycoprotein (PS beta G) is a polymorphic placental protein which shows strong sequence similarity with the oncofetal protein, carcinoembryonic antigen. To better understand the role of PS beta G in pregnancy, we examined its synthesis and regulation in placental fibroblasts, which had been shown to express the PS beta G gene. The major placental PS beta G is a 72-kDa glycoprotein, while the major fibroblast PS beta G is a 62-kDa species. Administration of sodium butyrate to these fibroblasts slightly stimulated the synthesis of the 62-kDa species but markedly increased the production of two additional PS beta Gs of 72 and 48 kDa. The similarity between the PS beta Gs synthesized by butyrate-treated fibroblasts and human placenta was confirmed by cell-free protein synthesis. Poly(A)+ RNA from butyrate-treated fibroblasts and placenta directed the synthesis of two polypeptides of 48 and 36 kDa, which form the polypeptide backbone of the 72- and 48-kDa glycoproteins. Moreover, the predicted molecular weights of PS beta Gs encoded by the two types of PS beta G cDNA clones were 48,000 and 36,000. Most PS beta G cDNAs identified to date, including the three cDNAs (PSG16, PSG93, and PSG95) isolated in this laboratory, share strong sequence similarity at the 5' region (designated PSG-5') but differ in sequences at their 3' regions. The PSG-5', PSG93-specific, PSG16/PSG93-3', and PSG95-3' probes, which identify the majority of PS beta G mRNAs, hybridized with three PS beta G mRNAs of 2.3, 2.2, and 1.7 kilobases from placental fibroblasts. Butyrate increased the steady-state levels of all three mRNAs. Ribonuclease protection analysis showed that butyrate increased the PS beta G mRNAs containing the PSG-5' or PSG93-specific sequence to approximately 20% of human placental levels. However, unlike human term placenta, which predominantly expressed PS beta G mRNAs with 3'-sequences similar to PSG16/PSG93, the butyrate-treated fibroblasts expressed roughly equal levels of PS beta G mRNAs with the PSG16/PSG93-3' and PSG95-3' ends. All PS beta G cDNAs identified encode proteins with distinct carboxyl termini, suggesting that the composition of the 72-kDa species in placenta and butyrate-treated fibroblasts is likely to be different. Placental fibroblasts provide a unique model for the study of the mechanisms responsible for the differential expression of the PS beta G gene.

[Purification and chemical characterization of pregnancy-specific beta 1-glycoprotein (SP1)].

A pregnancy-specific beta 1-glycoprotein (SP1) was purified from human placenta using either an ion-exchange column [method I] or an anti-SP1 antibody immunoadsorbent column [method II]. The yields were 5.2% with a total of 1.2 mg per placenta [method I], and 9. 4% with a total of 2.2 mg per placenta [method II], respectively. The physicochemical properties (molecular weight, sedimentation coefficient, amino acid composition etc.) of SP1 purified from human placenta by these methods are essentially the same as those of typical SP1. It is thus apparent from the present results that these SP1 from pregnant serum and human placenta cannot be distinguished immunologically and biochemically.

[The hydrophobic properties of beta-glycoprotein from the blood serum of pregnant rats]. / Gidrofobnye svoistva beta-glikoproteina syvorotki krovi beremennykh krys.

During immunoelectrophoresis in the presence of tween-80, triton X-100 and ammonium sulfate blood serum beta-glycoprotein of pregnant rats migrated along with beta-globulins as a main single band; its minor components in zones of alpha- and gamma-globulins were not detected. beta-glycoprotein was completely absorbed by phenyl sepharose in the absence of ligand as well as when the spacer arm for phenyl group was short. When the phenyl group was linked with the template through a long spacer arm, three froms of beta-glycoprotein with different immunoelectrophoretic mobility were detected after absorbtion with phenyl sepharose. Hence, beta-glycoprotein is hydrophobic and is represented by alpha-, beta- and gamma-forms in blood plasma of pregnant rats.

Identification of proteins immunochemically related to human pregnancy-specific beta 1-glycoprotein in the rat placenta.

Human pregnancy-specific beta 1-glycoprotein (hPS beta G) consists of a set of glycoproteins present in placenta and maternal serum. This study characterized proteins in rat placenta that show immunological cross-reactivity with antisera to hPS beta G. Immunocytochemical studies using two independent preparations of anti-hPS beta G showed intense specific staining within basophilic cytotrophoblast cells of the basal zone of the gestation day 15 rat placenta. In contrast, basophilic cytotrophoblasts located in the labyrinth did not stain. Subsequent experiments used gel electrophoresis and immunoblot analysis to compare PS beta G in human placenta and serum with immunoreactive proteins in rat placenta and serum. A set of two or three proteins was detected in human villous tissue and pregnancy serum with apparent mol wt (Mr) ranging from 54,000-76,000. In contrast rat placenta showed a major immunoreactive protein with 120,000 Mr, while rat serum contained bands of 48,000 64,000 and 69,000 Mr. Explant cultures of rat basal zone tissue secreted two [35S]methionine-labeled proteins that were immunoreactive, a major 120,000 Mr species and a minor 76,000 Mr form, with pI values of 4.6-5.5; tunicamycin inhibited the secretion of both species. Thus, a 120,000 Mr glycoprotein appears to be the major tissue and secreted form of rat PS beta G analog in day 15 placenta. Finally, the cytochemical localization of PS beta G-like proteins in rat placenta showed a progressive gestational shift from giant trophoblast cells in the parietal yolk sac placenta on day 12 to the basal zone cytotrophoblast cells by day 15. Data indicate that the pregnant rat may provide an animal model for investigation of the biological function of PS beta G during late gestation.

Human pregnancy-specific beta 1-glycoprotein: a new member of the carcinoembryonic antigen gene family.

The amino acid sequence of human pregnancy-specific beta 1-glycoprotein (PS beta G) as deduced from the cDNA sequence contains two repeated protein domains (1a and 2a) of 93 amino acids each. PS beta G shows strong homology to human carcinoembryonic antigen (CEA) at both nucleotide and amino acid levels. CEA contains eight domains including a N-terminal domain, three repeated domains (I, II, and III), each containing two subdomains (A and B), and a hydrophobic carboxyl-terminal domain. PS beta G contains a CEA-like N-terminal domain, two repeated domains similar to the A subdomains of CEA which is followed by a domain (1b) similar to the B subdomains of CEA, but lacks a hydrophobic carboxyl-terminal domain. The positions of the cysteine residues in each domain also are conserved, indicating that PS beta G and CEA are two members of the same gene family.

[Affinity of pregnant rat-specific beta 1-globulin for different lectins]. / Izuchenie affiniteta spetsificheskogo dlia beremennykh krys beta 1-globulina k razlichnym lektinam.

The pregnancy-specific beta 1-globulin (SBG) reacts with 10 out of 11 lectins which have affinity to monosaccharides (glucose, mannose, galactose and fucose) and acetylamino sugars. The affinity to ConA and PHA-P was the most pronounced while this protein did not react with the pea lectin. The SBG reactions are specific for every lectin (even with the identical carbohydrate specificity). Peculiarities of the SBG reactions with lectins made it possible to reveal a few forms of this protein which differ in the carbohydrate composition and conformation of carbohydrate radicals.

Interference of an alpha 2 component in immunological determinations of pregnancy-specific beta 1 glycoprotein in serum.

Pregnancy-specific beta 1 glycoprotein (SP1-beta) was purified from human retroplacental blood by sequential anion exchange chromatography, gel chromatography and affinity chromatography. The final preparation appeared to be electrophoretically and immunochemically pure and was in particular free from any component with alpha mobility. The preparation was used as immunogen in rabbits as well as tracer and standard for radioimmunoassay and for cross- and rocket-immunoelectrophoresis. It was shown that this radioimmunoassay procedure, allowed quantitative determination of SP1-beta glycoprotein without interference by the alpha component.

An immunochemical study of trophoblast-specific beta 1-glycoprotein and its fragments.

Trophoblast-specific beta 1-glycoprotein (TSG, SP-1) has been isolated from retroplacental blood serum by the usual techniques. Together with the purified TSG, an antigen (TSGfs) possessing partial immunochemical identity with TSG, has been isolated. This antigen, with molecular mass 25 KD, contains virtually no fucose nor neuraminic acid and differs from TSG in a lower content of arginine, tyrosine and aspartic acid. In addition, a low-molecular weight fragment of the TSG molecule (TSGhs), partially identical immunochemically with TSG and fully identical immunochemically with TSGfs, has been obtained by partial acid hydrolysis of TSG. The partial acid hydrolysis of TSG yields TSGhs and high-molecular weight fragments with molecular masses greater than 160 KD which also exhibit a partial identity with TSG. The results provide evidence for the occurrence of a labile linkage bonding the two parts of the TSG molecule which carry different immunochemical determinants.

Identification of an SP-1-like protein in non-pregnancy serum: isolation using a monoclonal antibody.

Human SP-1, a glycoprotein synthesized by the placenta during pregnancy, was shown to exist as polymers in maternal serum by a rapid passive transfer immunoblotting technique following conventional agarose electrophoresis. Moreover, the SP-1 polymers in serum were shown to dissociate into one main component upon treatment with 8 M urea prior to electrophoresis. However, an unexpected observation was the existence of an SP-1-like immunoreactive species in male serum with the sensitive immunoblotting technique. This SP-1-like protein in male serum had similar properties to its placentally derived counterpart in pregnancy serum, namely a propensity for complex formation and a reduced electrophoretic mobility following neuraminidase treatment. The relationship between the two SP-1 proteins was demonstrated by isolating them from their respective sera using an immobilized monoclonal antibody raised to purified SP-1 from pregnancy serum. Immunoblotting after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the existence of two major SP-1 species in pregnancy serum. These two SP-1 species had apparent molecular weights of 68,000 and 64,000. In addition, there were two minor bands at 35,000 and 32,000. These smaller SP-1 species did not appear to be subunits of the larger entities since they were detectable in the absence of reducing conditions. SDS-PAGE and immunoblotting showed that the immunoaffinity-purified SP-1 species from male and pregnancy sera had similar, but not identical, molecular weights.

[The purification of pregnancy specific beta 1-glycoprotein (SP1) from third trimester serum and the immunological identification of gamma-component of SP1].

This paper describes the SP1 purification procedures with high recovery from third trimester serum and the finding of a new SP1 antigen with gamma-mobility (tentatively called SP1-gamma). The serum was salted out at 40% saturation with ammonium sulfate and this was followed by Sephadex G-150, DEAE-cellulose, SP1-negative affinity chromatography and the mono-Q column of FPLC system. The anti-serum for the immunoadsorption techniques was made from the fractionated male serum. By these procedures, the final SP1 preparation was obtained in a yield of 16% and with a purity of 99%. The purified SP1 (SP1-beta) had beta-mobility and its molecular weight approximated 72,000 in SDS polyacryl-amide gel electrophoresis. The isoelectric points (pI) ranged between 3.80 to 4.55 using isoelectric focusing and the site at pI 4.15 was strongly stained. Another SP1, which passed through DEAE-cellulose with a low salt concentration, was detected in the gamma-region by means of immunoelectrophoresis using anti-SP1 serum. It was ascertained that the SP1-gamma was different from the SP1-beta treated with neuraminidase. The SP1-gamma precipitation fusing with SP1-beta could be observed even in pregnant serum by usual immunoelectrophoresis. The SP1-gamma preparation showed at least four pIs of 5.3, 5.5, 5.65 and 5.8.

[Use of Wheat Germ Lectin-Sepharose for isolating pregnancy-specific protein]. / Ispol'zovanie Wheat Germ Lectin-Sepharose dlia vydeleniia spetsificheskogo belka beremennosti.

Specific pregnancy protein was obtained from retroplacental blood serum by saline fractionation, affinity chromatography on Wheat germ Lectin-Sepharose, gel filtration on Sephadex G-200, hydrophobic chromatography on Phenyl-Sepharose, and ion exchange chromatography on DEAE-Sephacel. The purified preparation of specific pregnancy protein is highly suitable for preparing antisera and the development of immuno- and radioassays of the protein in question.