Isolation and characterization of eight pregnancy-associated glycoproteins present at high levels in the ovine placenta between day 60 and day 100 of gestation (original) (raw)
Related papers
Multiple Pregnancy-Associated Glycoproteins are Secreted by Day 100 Ovine Placental Tissue1
Biology of Reproduction, 1997
Pregnancy-associated glycoprotein (PAG)-1 (PAG1) and pregnancy-specific protein B are either identical or closely related antigens released by trophoblast binucleate cells of placentas of cattle. Sheep and other ruminants produce similar products. There is evidence, however, that these antigens, which are related structurally to the pepsinogens and other aspartic proteinases, are not single gene products but members of an extensive family. Here, the sequential use of ammonium sulfate precipitation and Sepharose blue, anion-exchange, and cation-exchange chromatographies, as well as isoelectric elution from a Mono P column, has allowed several PAG1-related molecules to be purified from the medium after culture of explants from Day 100 sheep placentas. Each of these PAGs cross-reacted to a varying extent with a panel of three different anti-PAG1 antisera. Four of them, all of which were major secretory products of the placenta, were subjected to amino-terminal microsequencing. Although each was related to ovine (ov) PAG1, none was identical. Reverse transcription-polymerase chain reaction was then used to amplify PAG1-related cDNA from Day 100 placental RNA. Seven novel full-length cDNA, all distinct from ovPAG1, were identified from 25 cDNA selected for sequencing. Only two of these (ovPAG3 and ovPAG7) encoded polypeptides identical in sequence at their inferred amino termini to one of the PAGs (ovPAG 65) purified from explant cultures. Even so, they were only 84% identical in overall sequence. The remaining five cDNA were unique. In situ hybridization analysis revealed that expression of ovPAG3 and ovPAG7, like that of ovPAG1, is confined to trophoblast binucleate cells. The data confirm that at Day 100 of pregnancy the ovine placenta produces many different PAGs, which differ considerably in sequence and immunological cross-reactivity.
Biology of Reproduction, 1998
Antigen(s) immunologically related to pregnancy-associated glycoproteins (PAGs) have previously been detected in the serum of pregnant goats. In this work, we describe a partial characterization of a family of PAGs isolated from the placenta of the goat. The procedure, monitored by RIA, included extraction of proteins at neutral pH, acidic, and ammonium sulfate precipitations; and gel filtration and ion exchange chromatographies. Immunoreactivity, initially located in the acidic supernatant and in the 40-80% ammonium sulfate fractions, was equally apportioned between the 0.04 and 0.08 M NaCI DEAE fractions. After further purification of both DEAE fractions, the preparations were subjected to oneand two-dimensional electrophoresis, and individual polypeptides were analyzed by amino acid sequencing.
Biology of Reproduction, 1996
Bovine and ovine pregnancy-associated glycoproteins-1 (PAG-1) are products of binucleate trophoblast cells and belong to the aspartic proteinase gene family. Estimates of their relative molecular masses have varied considerably, from 47 to 90 kDa, even though the mature polypeptide has been inferred to be no more than 330 amino acids in length and that the glycosylated recombinant form synthesized in Chinese hamster ovary (CHO) or COS-1 cells had an apparent mass of 46 kDa. To establish the relationships among the various molecular forms, metabolic labeling, immunoprecipitation, and electrophoretic analysis were used to follow the biosynthesis of ovine PAG-1 (ovPAG-1) in placental explants. In time-course studies, ovPAG-1 could first be detected within 10 min as a 70-kDa form within the tissue. With time, forms of intermediate (53-61 kDa) and low (47 kDa) molecular mass began to accumulate. The latter predominated in medium after 6 h labeling. Pulse chase studies established that the 70-kDa forms were the precursors of the smaller species. Inhibition of glycosylation with tunicamycin or treatment with N-glycosidase F confirmed that ovPAG-1 contained N-linked oligosaccharide chains, but that this carbohydrate accounted for only a relatively small fraction (8-10 kDa) of the apparent mass. Consecutive treatment with neuraminidase and O-glycanase also reduced the apparent molecular mass of the precursor by approximately 11 kDa. OvPAG-1 incorporated 32 p from [32Plorthophosphate into phosphoserine and phosphothreonine, but there was no incorporation of 35 S from [35S]sulfate. The basis of the differences in molecular mass between the precursor and the final products remains to be elucidated, but the differences seem likely to be due to some unusual form of posttranslational modification introduced in the binucleate cell. The results of the study appear to explain the disparate size values that have been reported for these placenta-derived proteins. Recently we identified cDNA for both bovine PAG-1 (boPAG-1) and ovine PAG-1 (ovPAG-1) [7] and were able to deduce the amino acid sequences of both proteins. Surprisingly, the PAG were structurally quite similar to pepsin and belonged to the aspartic proteinase gene family, a large grouping that includes chymosin, renin, and cathepsin D and E in addition to pepsin [16-22].
2007
Pregnancy diagnosis is an important part in reproduction management of ruminants. In the last years, a large polymorphic family of placenta-expressed proteins has been discovered in ruminant species and used for pregnancy diagnosis. Members of this family are named pregnancy-associated glycoproteins (PAG), being synthesized in the mono-and binucleate cells of the ruminant's trophectoderm. Part of them are released in the maternal blood circulation where they can be assayed by different laboratory techniques. Due to large variety of expressed molecules and to large variations in the post-translational processing of the PAG, different immuno-systems present different ability to quantify the PAG released in blood. The sensitivity (92 to 100%) and specificity of PAG radioimmunoassay when used for pregnancy diagnosis are very high. The assay of PAG can also bring very interesting information for researchers working in programs focused on the study of embryonic and fetal mortalities, as well as on embryo biotechnology (ET, FIV, clonage), animal nutrition, or infections diseases resulting in pathologies affecting the pregnancy.
Biology of Reproduction, 2000
The pregnancy-associated glycoproteins (PAG) constitute a large family of recently duplicated genes. They show structural resemblance to pepsin and related aspartic proteinases. A total of 21 bovine (bo) PAG and 9 ovine (ov) PAG cDNA have been identified. Phylogenetic analysis indicated that the PAG are divided into two main groupings that accurately reflect their tissue expression, as determined by in situ hybridization. In the first pattern, represented by ovPAG-2 and boPAG-2,-8,-10, and-11 (where the numbering is arbitrary and reflects order of discovery within species), expression occurred throughout the outer epithelial layer of the placenta (trophectoderm). The second pattern was predominant localization to binucleate cells. Ribonuclease protection assays, which allow discrimination between closely related transcripts, have shown that the expression of PAG varies in a temporal manner over pregnancy. Of those bovine PAG expressed predominantly in binucleate cells, boPAG-1,-6, and-7 are expressed weakly, if at all, by Day 25 placenta, but are present at the middle and end of pregnancy. Others, such as bo-PAG-4,-5, and-9, are expressed at Day 25 and at earlier stages. Although not among the earliest PAG produced by the trophoblast, boPAG-1 has been used for pregnancy diagnosis, particularly in dairy cows, where there is a major need for a sensitive method capable of detecting pregnancy within 1 mo of conception. It seems likely that some of the newly discovered PAG will be better candidates than PAG-1 for pregnancy diagnosis.
Theriogenology, 2003
Pregnancy-associated glycoproteins (PAGs) are antigens synthesized in the super®cial layers of the ruminant trophoblast. Initially, they were identi®ed either as proteins released into the maternal bloodstream (where they have applications in pregnancy diagnosis) (PAG1) or as molecules binding to the LH receptor (PAG2). In this study, double radial immunodiffusion was used to test the ability of antisera raised against different PAG molecules (bovine, ovine and caprine) to react with placental extracts from nonruminants (rabbit, cat, mouse, pig, and wild pig) and ruminants (cow, ewe, and goat). Placental extracts from all nonruminants tested except rabbit reacted with anti bovine PAG2 (anti-boPAG2). Extracts of ruminant placentas reacted with different antisera, con®rming the expression of various PAG molecules. According to the time at which the placentas were collected (early or middle pregnancy), the reaction differed as regards the thickness, position, and number of precipitation lines, suggesting that PAG expression varies as pregnancy progresses. Bos indicus and Bos taurus placental extracts exhibited different reactions with anti-boPAG2: a single precipitation line in the former case and two lines in the latter. This suggests differential expression of boPAG2 related glycoproteins in these two subspecies.
Biology of Reproduction, 1994
The pregnancy-associated glycoproteins (PAG 1) that appear in the maternal serum of cattle and sheep soon after implantation are apparently inactive members of the aspartic proteinase family. Here we describe the isolation of a highly abundant cDNA (PAG 2 cDNA) that represents a second member of this gene family which is structurally related to bovine PAG 1, ovine PAG 1, and pepsin (58%, 58%, and 51% amino acid sequence identity, respectively). The bovine PAG 2 cDNA was identified in two ways. First, the bovine placental library was screened under relatively nonstringent conditions with an ovine PAG 1 cDNA. The second fortuitous approach employed immunoscreening with an antiserum raised against a partially purified factor that competed with bovine LH for binding to the LH receptor on the CL of the ovary. The full-length cDNA (1258 bp) codes for a polypeptide of 376 amino acids. Bovine PAG 2, unlike bovine PAG 1, has a catalytic center with a consensus sequence of amino acids. Its mRNA is expressed in fetal placenta but not in other fetal organs, and is localized to both the mononucleate and binucleate cells of the trophectoderm, whereas PAG 1 is expressed only in binucleate cells. PAG 2 is synthesized by placental explants as a 70-kDa glycoprotein that is processed to several smaller molecules. Western blot analysis of culture media developed with epitopeselected antibodies to PAG 2 reveals several bands ranging in apparent M, from 31 000-70 000, which correspond in size to the polypeptides present in the preparation used for immunization. The function of PAG 2 remains unclear, but it could represent one of the poorly characterized gonadotropin-like factors described in placental extracts of cattle and sheep.
Altered secretion of pregnancy-associated glycoproteins during gestation in bovine somatic clones
Theriogenology, 2011
Somatic nuclear transfer (NT) in cattle is often accompanied by severe placental anomalies, hypertrophy, and hydrallantois, which induce a high rate of pregnancy losses throughout gestation. These placental deficits are associated with an abnormal increase of the maternal plasma levels of pregnancy-associated glycoprotein (PAG), produced by the trophoblastic binucleate cells (BNC) of the placenta. The objective of this study was to analyze the origin of the abnormally elevated PAG concentrations in the peripheral circulation of NT recipients during pathological pregnancies. Concentrations of PAG were measured both in maternal blood, in chorionic and cotyledonary tissular extracts from control recipients (after artificial insemination, AI, or in vitro fertilization, IVF) and clone recipients on Day 32, Day 62, and during the third trimester of gestation. Three different radioimmunoassay (RIA) systems were used. One homologous RIA for PSP60, similar to bovine PAG-1 (PAG 67kDa ), and two heterologous RIA with PAG 67kDa as standard and tracer, and antisera anti-caprine PAG (AS#706 and AS#708). Circulating and tissular concentrations of bovine placental lactogen (bPL), a glycoprotein also produced by BNC, were determined by RIA at the same stages. The number of BNC in the placental tissues was determined by cell counting after immunostaining with anti PSP60 antibody on tissue sections from control and NT pregnancies. Maternal plasma PAG concentrations were not different among groups on Day 32, but they were significantly higher in NT than in control pregnancies on Day 62 with all three RIA and during the third trimester with two RIA (RIA-PSP60 and RIA with AS#708). Circulating bPL concentrations were undetectable on Days 32 and 62 and were not different in the third trimester between NT and control pregnancies. Tissular amounts of PAG on total proteins were not different between the two groups at all stages studied. No difference was determined in the percentage of PSP60-positive BNC in placental tissues between controls and NT on Day 62 and during the third trimester of pregnancy. Western blots of tissular extracts from placenta showed no major molecular weight changes of PAG in NT pregnancies compared to controls. No differences in maternal circulation concentrations or tissular content of bPL were observed between control and NT pregnancies. In conclusion, the specific increase of PAG in maternal plasma concentrations during abnormal NT pregnancies do not result from a higher proportion of BNC, or an increased protein expression of PAG and could be due to changes in the composition of terminal glycosylation which result into a clearance decrease of PAG from the circulation.
Biology of Reproduction, 1992
A bovine pregnancy-associated glycoprotem (bPAG) of 67 kDa has previously been isolated from bovine fetal cotyledons. The objective of this study was to determine the cytological localization of that protein in the placentomes and possibly the cells responsible for its production. Highly specific antisera raised against pure bPAG were used to demonstrate the cellular localization of the protein in bovine placentomes by light and electron microscopic techniques. Strong immunostaining was observed exclusively in the cytoplasm of large binucleate cells present predominantly in fetal cotyledonary tissue (villi). Some smaller weakly immunostalned cells were also present in caruncular epitheium. By ultrastructural immunogold procedures, the protein was detected only within amorphous cytoplasmic granules. Granules of identical size, but weakly labeled, were found on the maternal side. All cells containing labeled granules were binucleate. These results suggest that bPAG is probably synthesized by trophoblast binucleate cells and stored in granules prior to delivery into the maternal circulation after cell migration.
Biology of Reproduction, 1995
Pregnancy-associated glycoproteins (PAG) are members of the aspartyl proteinase gene family that were initially identified in cattle (bPAG) and sheep (oPAG) as placenta-specific antigens in maternal blood. The objective of this study was to determine whether PAG are expressed in pig trophoblast. A porcine conceptus cDNA library was screened with 32P-labeled ovine and bovine PAG cDNA. Of the-104 plaques that were initially screened, a very high number (-5.3%) were positive. Two distinct types were identified, and full-length clones representing each type (1371 bp, pPAG1; 1378 bp, pPAG2) were fully sequenced in both directions. Their open reading frames coded polypeptides of 389 and 387 amino acids, respectively, including 15 amino acid signal peptides. Each had several potential sites for Nglycosylation. Both were members of the aspartic proteinase gene family, with-50% amino acid sequence identity to porcine pepsinogen and 64% to each other. They were only distantly related to PAG of ruminant species (53% and 49% identity in amino acid sequence to oPAG1 and bPAG1, respectively). Interestingly, pPAG1 had amino acid substitutions within its catalytic center (Gly-Ala 8 , domain 1; Thr-Ser 26 3 , Thr-*Ser28 5 , Ser-Ala 266 , domain 2) that together were likely to render it enzymatically inactive, whereas pPAG2 retained sequences identical to pepsin in these regions. Western blotting of secretory products of porcine trophoblast with anti-oPAG1 and anti-bPAG2 antisera indicated that pPAG, like PAG from ruminants, had an unexpectedly high Mr (-70 000). Northern blot analysis revealed abundant mRNA for both pPAG (-1.7 kb) as early as Day 15, which persisted throughout pregnancy. In situ hybridization localized pPAG mRNA to chorionic cells tightly adherent to the maternal luminal epithelium within the deep folds of endometrium. In conclusion, PAG are not restricted to species with a synepitheliochorial placenta, and their function may not depend upon their potential for peptide bond cleavage.