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.

Pregnancy-Specific ß1-Glycoproteins: Combined Biomarker Roles, Structure/Function Relationships and Implications for Drug Design.

BACKGROUND: Pregnancy specific ß1-glycoproteins (PSGs) have long been recognized as trophoblast quality and embryo viability markers. However, biological roles of PSGs remain obscure, and structure/function relationships to other feto-placental proteins as well as implications for drug design have not been reviewed. This review summarizes and discusses advances in 45-year studies of PSGs with focus on the latest achievements and the challenges for future investigations. METHODS: Literature search was performed to review the majority of recent PSG studies with emphasis on usage of high-throughput integrated proteomic profiling technologies, systems biology and bioinformatics approaches that enhance novel biomarker and drug target discovery as well as protein structure/activity analysis. RESULTS: Clinical significance and screening performance improved when PSG measurements were combined with those of other placenta-derived proteins: hCG, hPL, PAPP-A, and proMBP. Nevertheless, analysis of protein co-expression and co-localization data and the involvement of PSGs in protein interaction networks are being introduced to discover novel, specific and high-sensitive, gestational/cancer biomarkers. Despite biological roles of PSGs are not fully understood, there are evidences of that they exhibit immunomodulatory, antiinflammatory and proangiogenic effects. Investigation of structure/function relationships showed that PSGs may function in cooperative/coordinated manner with numerous regulatory proteins including alpha-fetoprotein and transforming growth factors-ß; this is provided by the presence of conserved short linear motifs (SLiMs) such as RGD, PXXP and AFP14-20-like (YXCX) ones. CONCLUSION: PSG-derived peptides may be used as a rationale to design novel drugs that mimic SLiMs involved in protein-protein interactions to inhibit domain-motif binding and to block cell signaling, and/or exert immunomodulatory, anti-inflammatory and proangiogenic effects.

Recombinant N-Domain of Pregnancy-Specific Glycoprotein from E. coli Cells: Analysis of the Spectrum of Polyclonal Antibodies.

We studied antibody spectrum in antisera to IgG-like recombinant N-domain of pregnancyspecific glycoprotein-1 (rPSG-N) from E. coli cells. In three experimental series, the fraction of IgG antibodies from anti-rPSG-N sera was immobilized on 3 immunoadsorbents: by polymerization with glutaraldehyde, on glutaraldehyde activated biogel P-300, and on commercial CNBr-activated 4B sepharose. Retroplacental serum was incubated with immobilized antibodies to rPSG1-N, protein was eluted and tested in the precipitation test in standard test systems with PSG1, IgG, and human serum albumin. Three proteins were eluted from all 3 immunoadsorbents: PSG1, IgG, and human serum albumin, which demonstrated the spectrum of antibodies to 3 proteins present also in natural serum PSG1 complex. The proportions of PSG1 and IgG obtained in these experiments were similar to those in natural serum PSG1 complex, while the level of human serum albumin was significantly higher in natural PSG1 complex. Thus, we failed to obtain PSG1 monoprotein free from IgG and human serum albumin. Antigenic mosaicism of the polypeptide chain of IgG-like rPSG1-N relative to the antigenic polyvalence of the complex of three proteins present in bioactive preparation of natural serum PSG1 was discussed.

Correlation between biological activity and conformational dynamics properties of tetra- and pentapeptides derived from fetoplacental proteins.

In this work, using molecular dynamics simulation, we study conformational and dynamic properties of biologically active penta- and tetrapeptides derived from fetoplacental proteins such as alpha-fetoprotein, pregnancy specific ß1-glycoprotein, and carcinoembryonic antigen. Existence of correlation between flexibility of peptide backbone and biological activity of the investigated peptides was shown. It was also demonstrated that flexibility of peptide backbone depends not only on its length, but also on the presence of reactive functional groups in amino acid side chains that participate in intramolecular interactions. Peptides that demonstrate similar biological effects in regulation of proliferation of lymphocytes and expression of differentiation antigens on their surface (LDSYQCT, PYECE, YECE, and YVCE) are characterized by rigidity of their peptide backbone. Increased backbone flexibility in peptides PYQCE, YQCE, SYKCE, YQCT, YQCS, YVCS, YACS, and YACE is correlated with decreased biological activity. Conformational mobility of amino acid residues does not depend on physicochemical properties only, but also on intramolecular interactions. So, evolutionary restrictions should exist to maintain such interactions in the environment of functionally important sites.

Conservation of pregnancy-specific glycoprotein (PSG) N domains following independent expansions of the gene families in rodents and primates.

BACKGROUND: Rodent and primate pregnancy-specific glycoprotein (PSG) gene families have expanded independently from a common ancestor and are expressed virtually exclusively in placental trophoblasts. However, within each species, it is unknown whether multiple paralogs have been selected for diversification of function, or for increased dosage of monofunctional PSG. We analysed the evolution of the mouse PSG sequences, and compared them to rat, human and baboon PSGs to attempt to understand the evolution of this complex gene family. RESULTS: Phylogenetic tree analyses indicate that the primate N domains and the rodent N1 domains exhibit a higher degree of conservation than that observed in a comparison of the mouse N1 and N2 domains, or mouse N1 and N3 domains. Compared to human and baboon PSG N domain exons, mouse and rat PSG N domain exons have undergone less sequence homogenisation. The high non-synonymous substitution rates observed in the CFG face of the mouse N1 domain, within a context of overall conservation, suggests divergence of function of mouse PSGs. The rat PSG family appears to have undergone less expansion than the mouse, exhibits lower divergence rates and increased sequence homogenisation in the CFG face of the N1 domain. In contrast to most primate PSG N domains, rodent PSG N1 domains do not contain an RGD tri-peptide motif, but do contain RGD-like sequences, which are not conserved in rodent N2 and N3 domains. CONCLUSION: Relative conservation of primate N domains and rodent N1 domains suggests that, despite independent gene family expansions and structural diversification, mouse and human PSGs retain conserved functions. Human PSG gene family expansion and homogenisation suggests that evolution occurred in a concerted manner that maintains similar functions of PSGs, whilst increasing gene dosage of the family as a whole. In the mouse, gene family expansion, coupled with local diversification of the CFG face, suggests selection both for increased gene dosage and diversification of function. Partial conservation of RGD and RGD-like tri-peptides in primate and rodent N and N1 domains, respectively, supports a role for these motifs in PSG function.

Structure and evolution of the mouse pregnancy-specific glycoprotein (Psg) gene locus.

BACKGROUND: The pregnancy-specific glycoprotein (Psg) genes encode proteins of unknown function, and are members of the carcinoembryonic antigen (Cea) gene family, which is a member of the immunoglobulin gene (Ig) superfamily. In rodents and primates, but not in artiodactyls (even-toed ungulates / hoofed mammals), there have been independent expansions of the Psg gene family, with all members expressed exclusively in placental trophoblast cells. For the mouse Psg genes, we sought to determine the genomic organisation of the locus, the expression profiles of the various family members, and the evolution of exon structure, to attempt to reconstruct the evolutionary history of this locus, and to determine whether expansion of the gene family has been driven by selection for increased gene dosage, or diversification of function. RESULTS: We collated the mouse Psg gene sequences currently in the public genome and expressed-sequence tag (EST) databases and used systematic BLAST searches to generate complete sequences for all known mouse Psg genes. We identified a novel family member, Psg31, which is similar to Psg30 but, uniquely amongst mouse Psg genes, has a duplicated N1 domain. We also identified a novel splice variant of Psg16 (bCEA). We show that Psg24 and Psg30 / Psg31 have independently undergone expansion of N-domain number. By mapping BAC, YAC and cosmid clones we described two clusters of Psg genes, which we linked and oriented using fluorescent in situ hybridisation (FISH). Comparison of our Psg locus map with the public mouse genome database indicates good agreement in overall structure and further elucidates gene order. Expression levels of Psg genes in placentas of different developmental stages revealed dramatic differences in the developmental expression profile of individual family members. CONCLUSION: We have combined existing information, and provide new information concerning the evolution of mouse Psg exon organization, the mouse Psg genomic locus structure, and the expression patterns of individual Psg genes. This information will facilitate functional studies of this complex gene family.

Evolutionary analysis of the multigene pregnancy-specific beta 1-glycoprotein family: separation of historical and nonhistorical signals.

The pregnancy-specific beta 1-glycoproteins (PSG) form a large family of closely related proteins. Using newly developed methods of sequence analysis, in combination with protein modeling, we provide a framework for investigating the evolution and biological function of genes like the PSG. Evolutionary trees, based on C-terminal sequence, group PSG genes in a manner consistent with their genomic organization. Trees constructed using the N-terminal domain sequences are unreliable as an indicator of phylogeny because of non-neutral processes of sequence change. During duplication of the PSG genes, evolutionary pressures have resulted in a gradient of constrained change across each gene. The N-terminal domains show a nonrandom pattern of amino acid substitutions clustered in the immunoglobulin complementarity-determining region (CDR)-like regions, which appear to be important in the function of the protein.

[Evaluation of trisomy 21 risk by serial determination of chorionic gonadotrophin hormone and alpha-fetoprotein: results of a national pilot study]. / Evaluation du risque de trisomie 21 par dosage sérique de l'hormone gonadotrophique chorionique et de l'alpha-foetoprotéine: résultats de l'enquête pilote nationale.

Previous studies, as early as 1984, have demonstrated the efficiency of maternal serum markers to screen for Down syndrome. These markers were AFP, hCG, oestriol and beta-1-glycoprotein. A pilot study was initiated in France in 1990 to evaluate these markers. It lasted from May 1990 until April 1991. 22,410 pregnancies were monitored in total, and 19,407 for women between 30 and 37 years of age. The pregnancy outcome was known for 20,151 cases (86.6%). Sensitivity and predictive value of the test was calculated on 17,362 dosages which outcome was known. The sensitivity based on hCG only was 59.4% (38/64) for trisomy 21 and positive predictive value 1.65% (38/2,307). This test is a better marker than maternal age. In the pilot study it induced a 13.2% rate of amniocentesis. If the risk was calculated using both hCG and AFP results, the performances of the test were better. At equal rate of amniocentesis, the double test increases the sensitivity (+9.5% at risk 1/200, +7% at risk 1/250, +14.4% at risk (1/350). The strategy of double dosages decreases the number of amniocentesis for a given sensitivity rate. This decrease is more spectacular for high levels of sensitivity. This pilot study confirms already published results. Maternal serum markers are the best tools, combined with maternal age to evaluate the risk of trisomy 21.

Pregnancy-associated plasma protein B (PAPP-B): isolation from human late pregnancy serum and biochemical characterization.

The pregnancy associated plasma protein B (PAPP-B) was first described by Lin et al. as a glycoprotein with a molecular weight of about 1,000 kD and a serum half life of less than 24 hours after delivery. We isolated PAPP-B from late pregnancy serum using liquid chromatography including HPLC. PAPP-B was found to be a glycoprotein with a molecular weight of 1,300 kD and to be an octadecamer of identical subunits (74 kD each). Cleavage of the carbohydrates with trifluoromethanesulfonic acid reduced the molecular mass to 65 kD (by SDS PAGE, 12.2%). Isoelectrofocusing and titration curves fixed the isoelectric point at 5.3. Spectroscopy measurements between 230 and 360 nm yielded a trp/tyr ratio of 1.1. Amino acid analysis showed that each subunit contained five methionines; this was confirmed by a CNBr cleavage experiment.

cDNA sequence of the pregnancy-specific beta 1-glycoprotein-11s (PSG-11s).

Four cDNA clones representing the human pregnancy-specific beta 1-glycoprotein-11 (PSG-11) gene have been characterised. All encoded a splice variant of the PSG-11 gene designated PSG-11s, which can encode a secreted protein of 426 amino acids, containing six potential N-linked glycosylation sites, with a domain structure L-N-AI-AII-BII-C. Minor differences between the four clones sequenced included a restriction site polymorphic for ApaI that may differentiate between alleles of the PSG-11 gene.

Distribution of SP1, immunoreactivity among different plasma proteins: real molecular heterogeneity or adsorption of SP1-beta to other plasma proteins?

Three SP1-containing factors from pooled term pregnancy sera were subjected to crossed immunoelectrophoresis. New patterns as far as electrophoretic mobilities and shapes of the immunoprecipitates were revealed. The appearance of an additional anodic radioimmunoassayable activity in agarose electrophoresis of mixed SP1-alpha and SP1-beta suggested a binding capacity of SP1-alpha for SP1-beta determinants. In the serum of a single patient at the third trimester of pregnancy we also found two SP1 variants, possessing little radioimmunological reactivity and with crossed immunoelectrophoretic characteristics quite different from those of the 'usual' alpha and beta SP1 forms. These results suggest that, in this particular case, the overall SP1 production cannot be evaluated by competitive binding assay and, that in general, SP1 is a complex antigen the heterogeneity of which can be determined following adsorption of some beta epitopes to another serum protein.

Pediatric germ cell and human chorionic gonadotropin-producing tumors. Clinical and laboratory features.

Germ cell tumors may cause various aberrations in pubertal development. In prepubertal boys, these tumors may secrete human chorionic gonadotropin, resulting in precocious puberty. Human chorionic gonadotropin and alpha-fetoprotein are both useful as germ cell tumor markers in the diagnosis and detection of recurrence. Pregnancy-specific beta 1-glycoprotein, another oncoplacental antigen, has been used as a tumor marker for trophoblastic neoplasms, but not previously for human chorionic gonadotropin-producing tumors associated with precocious puberty. Patients with germ cell tumors may also have abnormal karyotypes. Herein, we describe six male pediatric patients with germ cell tumors and pubertal derangements seen during an 8-year period. We confirm the high incidence of associated sexual precocity, the usefulness of alpha-fetoprotein, human chorionic gonadotropin, and pregnancy-specific beta 1-glycoprotein as tumor markers in the diagnosis and follow-up of these patients, and the occurrence of sex chromosomal abnormalities.

Characterization of new members of the pregnancy-specific beta 1-glycoprotein family.

Three cDNAs encoding members of the pregnancy-specific beta 1-glycoprotein (PSG) family were isolated from human term placental cDNA library. All three cDNAs encode proteins with similar domain structure. There is a leader sequence of 34 amino acids followed by an N-domain of 109 amino acids. Immediately after the N-domain are one or two copies of a repeating A-domain of 93 amino acids, a B-domain of 85 amino acids and a C-domain of variable size. The proteins are highly hydrophilic. However, one of them has an 81-amino acid C-domain which is very hydrophobic and could potentially serve as a membrane attachment site. The putative cell-cell recognition tripeptide, Arg-Gly-Asp, is present in the N-domain of two of the proteins. Partial sequence of one of the cDNAs has been found in HeLa cells while cDNAs highly homologous to two of the cDNAs have been found in the fetal liver. Functional roles of the PSG proteins basing on their structure are proposed.

Placental function test, clinical and biochemical parameters in diabetic pregnancy complicated by retinopathy.

The pregnancy in specific-beta 1-glycoprotein (SP1) has been characterized as a beta 1 electrophoretic mobile glycoprotein with a molecular weight of 90,000 daltons. SP1 is known to be synthesized by the trophoblast. The measurement of this protein has been shown to be useful as a placental function test. At present, we have compared maternal SP1 serum levels in diabetic pregnancies between White classes A to D on the one hand and R, F on the other. A total of 37 uncomplicated pregnancies in healthy women and 32 of insulin-dependent pregnant diabetic women were examined between completed gestational weeks 8 and 41. In the diabetic group there were eleven women with diabetic retinopathy. Maternal SP1 serum levels were estimated by single radial immunodiffusion using a monospecific antiserum. In the results were integrated maternal and neonatal data such as glycemic control, glycosylated hemoglobin and insulin requirements. In each group there was a significant rise in maternal SP1 serum values in the second and the third trimester, when compared with values in the first trimester (p less than 0.01). Between the 34th and the 37th gestational week we found significantly lower SP1 values (p less than 0.05) in the retinopathic group (104.2 +/- 28.7 mg/l) in comparison with the control group (149.9 +/- 61.0 mg/l) and non-retinopathic group (139.1 +/- 41.7 mg/l).