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.

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.

Purification of pregnancy-specific beta 1-glycoprotein with a monoclonal antibody immunoadsorbent.

Hybrid cell lines secreting monoclonal antibodies against pregnancy-specific beta 1-glycoprotein (SP1) were produced by fusion of a mouse myeloma cell line with spleen cells from BALB/c mice immunized with purified placental SP1. The clones were further grown intraperitoneally in mice, and the ascites fluids contained anti-SP1 antibodies in high titers. One of the monoclonal antibodies was coupled to cyanogen-bromide-activated Sepharose and used for affinity chromatography of SP1 using late pregnancy serum as starting material. The purification of SP1 by means of immunoadsorption utilizing monoclonal antibodies offers remarkable advantages compared with the purification procedures used so far.

Observations on the separation of SP1 alpha and SP1 beta by affinity chromatography.

SP1 antigens in late pregnancy blood, in placental extracts and in acidified plasma have been purified on positive affinity chromatography columns with immobilized SP1 antiserum and on negative affinity columns with immobilized antihuman non-pregnancy serum. Both SP1 alpha and SP1 beta are bound by the positive column but only SP1 alpha is bound by the negative column. This is adduced as further support for the view that SP1 alpha is formed by the combination of SP1 beta with a protein present in non-pregnancy serum.

The demonstration of two pregnancy-associated proteins with SP1 determinants in placental extracts.

The SP1 content of the placenta has been examined. It was found that, as in pregnancy serum, two variants of the protein existed in the placenta. The smaller molecule, with a beta 1 electrophoretic mobility, is readily extractable with phosphate buffered saline. The second larger molecule is more tightly bound in the placenta and requires Triton X-100 to be solubilized. The hypothesis is put forward that the larger alpha variant is the precursor form which is related more irregularly and in a smaller amount from the placenta into the maternal circulation.

Molecular heterogeneity of pregnancy-specific beta 1-glycoprotein (SP1).

Ion-exchange chromatography has been used to isolate three separate forms of Pregnancy-Specific beta 1-Glycoprotein (SP1) from pooled, late-pregnancy human sera. Each of the three components was immunochemically identifiable as SP1, yet distinguishable from each other on the basis of rocket morphology resulting from rocket immunoelectrophoresis (RIE). The elution profile from DEAE Sephacel indicated that each component has an isoelectric point below pH 8.6, but suggested a different charge for each molecular species. Crossed immunoelectrophoresis (XIE) of pooled, late pregnancy serum suggested the presence of each component prior to physico-chemical treatment and showed the components to be characterised by alpha, beta or gamma electrophoretic mobility. The differences in rocket morphology suggested differing affinities for the antiserum, and a difference in molecular size, which was confirmed by gel filtration.

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.

Heterogeneity of the pregnancy-specific beta 1-glycoprotein (SP1).

The pregnancy-specific beta 1-glycoprotein (SP1) in pregnant serum fractionated by ammonium sulfate precipitation and anion exchange chromatography was investigated by various electrophoretic methods. From these investigations it appears that in addition to the major component SP1(beta) three other components are present, termed SP1(gamma), SP1 (alpha 2(2)) and SP1 (alpha 2(1) on the basis of their electrophoretic mobilities. The isoelectric point (pI), estimated by isoelectric focusing, and the relative molecular mass (Mr), estimated by Sephadex G-200 gel filtration chromatography are for SP1(gamma): pI 2.9--4.9, Mr approximately 80000; SP1(beta): pI 2.5--4.5, Mr approximately 80000; SP1(alpha 2(2)): pI less than or equal to 6, Mr greater than or equal to 200000; SP1(alpha 2(1)): pI less than 3, Mr approximately 30000. Based on the interaction with phenyl-Sepharose and concanavalin A all four species of SP1 are amphiphilic glycoproteins.

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.

[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.

[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.

[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.

[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.

Pregnancy-specific beta 1-glycoprotein (SP1). Purification and partial characterization.

The pregnancy-specific beta 1-glycoprotein, SP1, was purified to homogeneity from placental extract and from serum using immunoadsorbent techniques. The isolated SP1 had a molecular weight of 90,000-110,000 daltons and an amino acid and amino sugar composition typical of an acidic glycoprotein. The availability of purified SP1 will be of value in the further evaluation of the role and importance of SP1 in malignancies as well as in normal development.

[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.

Immunoadsorbent isolation of pregnancy-specific beta 1-glycoprotein from maternal serum.

A three-step procedure for the purification of pregnancy-specific beta 1-glycoprotein (PS beta 1G) on a milligram scale from maternal serum has been developed. The principal purification was achieved by the use of an immunoadsorbent and the remaining impurities were removed by hydroxylapatite chromatography and negative affinity chromatography. The overall procedure resulted in the purification of approximately 10 mg of PS beta 1G which represented about 21% of PS beta 1G in 300 mL, of serum. The PS beta 1G was of high purity as shown by analytical polyacrylamide gel electrophoresis, sodium dodecyl sulfate--polyacrylamide gel electrophoresis, and immunochemical tests. Experiments by immunoelectrophoresis and gel chromatography indicate that the electrophoretic mobility and relative mass of the purified PS beta 1G are very similar to those of the native serum protein. Structural analysis of PS beta 1G suggests that it is composed of two identical subunit chains bonded noncovalently. However, a trimeric structure for PS beta 1G cannot be ruled out based on the uncertainty of relative mass estimates by gel chromatography in nondenaturing solvent. The anomalous characteristics of a previous purified polymeric form of PS beta 1G (PS beta 1G-I) are discussed in relation to the new findings presented here.

[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.

Separation of two pregnancy-associated proteins with SP1 determinants and the conversion of SP1 beta to SP1 alpha.

The alpha and beta forms of the pregnancy-associated protein, SP1 which have been previously identified in late pregnancy serum were found to be also present in the placenta. The alpha form was somewhat more difficult to extract, requiring the use of detergents in the extracting buffer. The two variants can be separated by ion exchange chromatography or by gel filtration and distinguished by the different shapes of the precipitation rockets on immunoelectrophoresis. The alpha variant appears to be the larger molecule on gel filtration. When solutions of SP1 are acidified to pH 2.5 the beta form is rapidly converted to the alpha form and the latter is slowly destroyed. It is suggested that SP1 alpha represents the combination of SP1 beta with some other protein present in the placenta and in the serum.