Aptamers based sensing of pregnancy associated glycoproteins (PAG) of bovine for early pregnancy detection.
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ABSTRACT:
SUBMITTER: Yadav ML
PROVIDER: S-EPMC8636505 | biostudies-literature | 2021 Dec
REPOSITORIES: biostudies-literature
Publications
Aptamers based sensing of pregnancy associated glycoproteins (PAG) of bovine for early pregnancy detection.
Yadav Munna Lal ML Parashar Abhishek A Mahanandia Nimai Charan NC Bhushan Vanya V Kumar Sudarshan S Mohanty Ashok Kumar AK
Scientific reports 20211201 1
Tosyl activated magnetic beads were used for aptamer selection against PAG- 7 and 18 proteins of bovine origin. PAG proteins were immobilized on beads with further addition of biotin tagged aptamer library. The recognition of aptamers with PAG was identified by ST-HRP based approach which was colorimetric in nature. The selected aptamers were sequenced and at the same time several new aptamers were identified. Later M-fold structure and G-quadruplex score of aptamers were analyzed for their sele ...[more]
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Project description:Bovine pregnancy-associated glycoproteins (boPAGs) are extensively glycosylated secretory proteins of trophoblast cells. Roughly 20 different boPAG members are known but their distribution patterns and degree of glycosylation during pregnancy are not well characterized. The objective of the present study was the development of a parallel reaction monitoring-based assay for the profiling of different boPAGs during pregnancy and after gestation. Furthermore, we investigated the effects of N-glycosylation on our analytical results. BoPAGs were purified from cotyledons of four different pregnancy stages. The assay detects 25 proteotypic peptides from 18 boPAGs in a single run. The highest abundances were found for boPAG 1 in both, glycosylated and deglycosylated samples. Strongest effects of glycosylation were detected during mid and late pregnancy as well as in afterbirth samples. Furthermore, we identified different boPAG-clusters based on the observed relative protein abundances between glycosylated and deglycosylated samples. A linkage between the impact of glycosylation and potential N-glycosylation sites or phylogenetic relation was not detected. In conclusion, the newly developed parallel reaction monitoring-based assay enables for the first time a comprehensive semi-quantitative profiling of 18 different boPAGs during pregnancy and post-partum on protein level, thereby investigating the influence of glycosylation. The results of this study provide new and important starting points to address further research on boPAGs to better understand their physiological role during pregnancy and for the development of new pregnancy detection tests.
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Project description:The pregnancy-associated glycoproteins (PAGs) represent a complex group of putative aspartic peptidases expressed exclusively in the placentas of species in the Artiodactyla order. The ruminant PAGs segregate into two classes: the 'ancient' and 'modern' PAGs. Some of the modern PAGs possess alterations in the catalytic center that are predicted to preclude their ability to act as peptidases. The ancient ruminant PAGs in contrast are thought to be peptidases, although no proteolytic activity has been described for these members. The aim of the present study was to investigate (1) if the ancient bovine PAGs (PAG-2 and PAG-12) have proteolytic activity, and (2) if there are any differences in activity between these two closely related members. Recombinant bovine PAG-2 and PAG-12 were expressed in a baculovirus expression system and the purified proteins were analyzed for proteolytic activity against a synthetic fluorescent cathepsin D/E substrate. Both proteins exhibited proteolytic activity with acidic pH optima. The k(cat)/K(m) for bovine PAG-2 was 2.7x10(5) m(-1) s(-1) and for boPAG-12 it was 6.8x10(4) m(-1) s(-1). The enzymes were inhibited by pepstatin A with a K(i) of 0.56 and 7.5 nm for boPAG-2 and boPAG-12, respectively. This is the first report describing proteolytic activity in PAGs from ruminant ungulates.
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Project description:Bovine pregnancy-associated glycoproteins (boPAGs) are extensively glycosylated secretory proteins of trophoblast cells. Roughly 20 different boPAG members are known but their distribution patterns and degree of glycosylation during pregnancy are not well characterized. The objective of the present study was the development of a parallel reaction monitoring-based assay for the profiling of different boPAGs during pregnancy and after gestation. Furthermore, we investigated the effects of N-glycosylation on our analytical results. BoPAGs were purified from cotyledons of four different pregnancy stages. The assay detects 25 proteotypic peptides from 18 boPAGs in a single run. The significantly highest protein abundances were found for boPAG 1 in both, glycosylated and deglycosylated samples. Strongest effects of glycosylation were detected during mid and late pregnancy as well as in afterbirth samples. Furthermore, we identified different boPAG-clusters based on the observed relative protein abundances between glycosylated and deglycosylated samples. A linkage between the impact of glycosylation and potential N-glycosylation sites or phylogenetic relation was not detected. In conclusion, the results of this study provide new starting points to address further research on boPAGs to better understand their physiological role during pregnancy.
2022-07-05 | PXD027383 | Pride
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2022-07-05 | PXD034108 | Pride
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| S-EPMC3842769 | biostudies-literature
Project description:BackgroundIn food production animals, especially cattle, the diagnosis of gestation is important because the timing of gestation directly affects the running of farms. Various methods have been used to detect gestation, but none of them are ideal because of problems with the timing of detection or the accuracy, simplicity, or cost of the method. A new method for detecting gestation, which involves assessing interferon-tau (IFNT)-stimulated gene expression in peripheral blood leukocytes (PBL), was recently proposed. PBL fractionation methods were used to examine whether the expression profiles of various PBL populations could be used as reliable diagnostic markers of bovine gestation.MethodsPBL were collected on days 0 (just before artificial insemination), 7, 14, 17, 21, and 28 of gestation. The gene expression levels of the PBL were assessed with microarray analysis and/or quantitative real-time reverse transcription (q) PCR. PBL fractions were collected by flow cytometry or density gradient cell separation using Histopaque 1083 or Ficoll-Conray solutions. The expression levels of four IFNT-stimulated genes, interferon-stimulated protein 15 kDa (ISG15), myxovirus-resistance (MX) 1 and 2, and 2'-5'-oligoadenylate synthetase (OAS1), were then analyzed in each fraction through day 28 of gestation using qPCR.ResultsMicroarray analysis detected 72 and 28 genes in whole PBL that were significantly higher on days 14 and 21 of gestation, respectively, than on day 0. The upregulated genes included IFNT-stimulated genes. The expression levels of these genes increased with the progression of gestation until day 21. In flow cytometry experiments, on day 14 the expression levels of all of the genes were significantly higher in the granulocyte fraction than in the other fractions. Their expression gradually decreased through day 28 of gestation. Strong correlations were observed between the expression levels of the four genes in the granulocyte fractions obtained with flow cytometry and with density gradient separation.ConclusionsThe expression profiles of ISG15, MX1, MX2, and OAS1 could be a useful diagnostic biomarker of bovine gestation. Assessing the expression levels of these genes in a granulocyte fraction obtained with density gradient separation is a practical way of detecting gestation in cows within three weeks of insemination.
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Project description:The pregnancy-associated glycoprotein-like family (PAG-L) is a large group of chorionic products, expressed in the pre-placental trophoblast and later in the post-implantational chorionic epithelium, and are involved in proper placenta development and embryo-maternal interaction in eutherians. This study describes identification of the PAG-L family in the genome of the Eurasian beaver (Castor fiber L.), named CfPAG-L. We identified 7657 bp of the CfPAG-L gDNA sequence (Acc. No. KX377932), encompassing nine exons (1-9) and eight introns (A-H). The length of the CfPAG-L exons (59-200 bp) was equivalently similar to the only known counterparts of bPAG1, bPAG2, and pPAG2. The length of the CfPAG-L introns ranged 288-1937 bp and was completely different from previously known PAG introns. The exonic CfPAG-L regions revealed 50.3-72.9% homology with equivalent segments of bPAG1 and pPAG2 structure. The intronic CfPAG-L regions alignments revealed a lack of homology. Within the entire CfPAG-L gene, 31 potential single nucleotide variants (SNV: 7 transversions and 24 transitions) were predicted. The identified exonic polymorphic loci did not affect the amino acid sequence of the CfPAG-L polypeptide precursor. This is the first report describing the CfPAG-L gene sequence, structural organization, and SNVs in the Eurasian beaver, one of the largest rodents.
| S-EPMC5561160 | biostudies-literature
Project description:Background: In food production animals, especially cattle, the diagnosis of gestation is an important issue because the timing of gestation has direct effects on the running of farms. Various methods have been used to detect gestation, but none of them are ideal because of problems with the timing of detection, or the accuracy, simplicity, or cost of the method, etc. A new method for detecting gestation, which involves assessing interferon-tau-related gene expression in peripheral blood leucocytes (PBL), was recently proposed. PBL fractionation methods were used to examine whether the expression profiles of various PBL populations could be used as reliable diagnostic markers of bovine gestation. Methods: PBL were collected on days 0 (just before artificial insemination), 7, 14, 17, 21, and 28 of gestation. The gene expression levels of the PBL were assessed with microarray analysis and/or quantitative real-time reverse transcription (q)-PCR. PBL fractions were collected with flow cytometry or density gradient cell separation using Histopaque 1083 or Ficoll-Conray solution. The expression levels of four interferon-tau-related genes, interferon-stimulated protein 15 kDa (ISG15), myxovirus-resistance (Mx) 1 and 2, and 2'-5'-oligoadenylate synthetase (OAS1), were then analysed in each fraction until day 28 of gestation using qPCR. Results: Miroarray analysis detected 72 and 28 genes significantly higher in whole PBL on days 14 and 21 of gestation compare to those on day 0, respectively. They involved interferon-tau responded genes. The expression levels of these genes increased with the progression of gestation. In the flow cytometry experiment, on day 14 all of the genes displayed significantly higher expression levels in the granulocyte fraction than the other fractions, and their expression levels gradually decreased until day 28 of gestation. Strong correlations were observed between the expression levels of the four genes in the granulocyte fractions obtained with flow cytometry and density gradient separation. Conclusions: The expression profile of the four abovementioned genes could be a useful diagnostic biomarker of bovine gestation. Assessing the expression levels of ISG15, Mx1, Mx2 and OAS1 in a granulocyte fraction obtained with density gradient separation is a practical way of detecting gestation in cows within three weeks of insemination.
2013-01-31 | GSE42894 | GEO
Project description:Background: In food production animals, especially cattle, the diagnosis of gestation is an important issue because the timing of gestation has direct effects on the running of farms. Various methods have been used to detect gestation, but none of them are ideal because of problems with the timing of detection, or the accuracy, simplicity, or cost of the method, etc. A new method for detecting gestation, which involves assessing interferon-tau-related gene expression in peripheral blood leucocytes (PBL), was recently proposed. PBL fractionation methods were used to examine whether the expression profiles of various PBL populations could be used as reliable diagnostic markers of bovine gestation. Methods: PBL were collected on days 0 (just before artificial insemination), 7, 14, 17, 21, and 28 of gestation. The gene expression levels of the PBL were assessed with microarray analysis and/or quantitative real-time reverse transcription (q)-PCR. PBL fractions were collected with flow cytometry or density gradient cell separation using Histopaque 1083 or Ficoll-Conray solution. The expression levels of four interferon-tau-related genes, interferon-stimulated protein 15 kDa (ISG15), myxovirus-resistance (Mx) 1 and 2, and 2'-5'-oligoadenylate synthetase (OAS1), were then analysed in each fraction until day 28 of gestation using qPCR. Results: Miroarray analysis detected 72 and 28 genes significantly higher in whole PBL on days 14 and 21 of gestation compare to those on day 0, respectively. They involved interferon-tau responded genes. The expression levels of these genes increased with the progression of gestation. In the flow cytometry experiment, on day 14 all of the genes displayed significantly higher expression levels in the granulocyte fraction than the other fractions, and their expression levels gradually decreased until day 28 of gestation. Strong correlations were observed between the expression levels of the four genes in the granulocyte fractions obtained with flow cytometry and density gradient separation. Conclusions: The expression profile of the four abovementioned genes could be a useful diagnostic biomarker of bovine gestation. Assessing the expression levels of ISG15, Mx1, Mx2 and OAS1 in a granulocyte fraction obtained with density gradient separation is a practical way of detecting gestation in cows within three weeks of insemination. Gene expression profiles were analyzed in the peripheral blood leucocytes at days 0 (n=5), 14 (n=3) and 21 (n=5) after artificial insemination in cow.
2013-01-31 | E-GEOD-42894 | biostudies-arrayexpress