2D DIGE analysis of maternal plasma for potential biomarkers of Down Syndrome

<p>Abstract</p> <p>Background</p> <p>Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.</p> <p>Results</p> <p>We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.</p> <p>Conclusions</p> <p>Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.</p

A new chapter in Rh research: Rh proteins are ammonium transporters

Occasionally, an original research paper has an unusually significant impact on a particular research field. Such a paper, published recently in Nature Genetics, describes the uncovering of the functional role of the Rh protein family - the proteins that express the Rh blood group antigens. Marini et al.1 demonstrate how two human Rh glycoproteins can correct ammonium transport deficiency in mutant yeast cells. Rh proteins are therefore ammonium transporters - a role that, in vertebrates, has remained previously uncharacterized. These data herald a new era in Rh protein research, beyond their role as blood group antigens, and into the characterization of ammonium transport mechanisms, notably in the kidney

Opposing effects of Protein Kinase A and C causes differential phosphatidyserine exposure in a CD47 receptor mediated erythrocyte apoptotic pathway

Erythrocyte apoptosis, like nucleated cell death, is characterised by phosphatidylserine exposure at the outer membrane leaflet. Our group has previously reported that ligation of a monoclonal antibody, BRIC 126 can mediate red cell apoptosis and subsequent PS exposure, through a CD47 receptor mediated pathway. We have also demonstrated that several membrane proteins are involved in this pathway and one such interaction is a direct protein: protein interaction between CD47 and protein 4.1R. Further studies have shown that red cells deficient in protein 4.1R undergo increased PS exposure in response to BRIC-126 than do normal cells, suggesting that protein 4.1R is critical to this pathway. In order to further elucidate the CD47 apoptotic pathway we have inhibited Protein Kinase A and Protein Kinase C whilst ligating CD47 with BRIC-126, as these kinases are known to interact with protein 4.1R at specific serine residues and are often implicated in cell signalling cascades, especially apoptosis. We have used flow cytometry in conjunction with an annexin V-FITC binding assay to compare mean percentage annexin V positive cells whilst inhibiting protein kinase A and protein kinase C using the cell permeable specific inhibitors Bisindolylmaleimide I, Go 6976 and KT 5720. Our findings suggest opposing effects of Protein kinase A and Protein Kinase C compared with control erythrocytes. We observed that PS exposure was decreased in cells with the specific cell permeable PKC inhibitors Bisindolylmaleimide I and Go 6976 present but increased in cells with the specific PKA cell permeable inhibitor KT5720 present. The increase in PS exposure when PKA inhibitor KT5720 is present is similar to the effect seen in individuals deficient in protein 4.1R. The difference in PS exposure between PKC inhibition and PKA inhibition suggests opposing effects of PKA and PKC during CD47 ligation and a possible interaction with protein 4.1R, this may also suggest that specific phosphorylation events on protein 4.1R are critical to the CD47 apoptotic pathway. Ongoing studies are investigating isoform specific inhibition of PKC and PKA along with 2D phosphoimmunoblots to look at changes in PI before and after ligation of BRIC 126

Modelling the human rhesus proteins: Implications for structure and function

The mammalian rhesus (Rh) proteins that carry the Rh blood group antigens of red blood cells are related to the ammonium channel (Amt) proteins found in both pro- and eukaryotes. However, despite their clinical importance the structure of the Rh antigens is presently unknown. We have constructed homology models of the human Rh proteins, RhD and RhAG using the structure of the Escherichia coli ammonia channel AmtB as a template, together with secondary structure predictions and the extensive available biochemical data for the Rh proteins. These models suggest that RhAG and the homologous non-erythrocyte Rhesus glycoproteins, RhBG and RhCG, have a very similar channel architecture to AmtB. By comparison, RhD and RhCE have a different arrangement of residues, indicating that if they function as ammonia channels at all, they must do so by a different mechanism. The E. coli AmtB protein is a homotrimer and our models provoke a reassessment of the widely accepted tetrameric model of the organisation of the erythrocyte Rh complex. A critical analysis of previously published data, together with sequencing yield data, lead us to suggest that the erythrocyte Rh complex could indeed also be trimeric. © 2005 Blackwell Publishing Ltd

Expression of phosphatidylserine (PS) on wild-type and Gerbich variant erythrocytes following glycophorin-C (GPC) ligation

Glycophorin-C (GPC) is a 40 kDa glycoprotein expressed on erythrocytes and is a receptor for the malarial parasite Plasmodium falciparum to invade these cells. A link between GPC binding (ligation) and phosphatidylserine (PS) expression on erythrocytes has been suggested by its appearance on P. falsiparum-infected erythrocytes. Phosphatidylserine expression has also been shown to be a marker of cellular death in a number of biological pathways including some in erythrocytes. Using Annexin V binding, we demonstrated that ligation of GPC with mouse mAb (BRIC-10) induced PS expression on normal erythrocytes. Phosphatidylserine exposure was prevented following tryptic digestion of intact erythrocytes. In addition, GPC variant phenotypes Yus (Δ exon 2) and Gerbich (Δ exon 3), which express a truncated extracellular domain, did not express PS following BRIC-10 binding, whereas PS was exposed on Lsa erythrocytes (duplication of exon 3). GPC ligation was also shown to result in a concomitant loss of erythrocyte viability in wild-type erythrocytes after 24 h in vitro. These results identify a potential pathway linking GPC to PS exposure on erythrocytes that may have a role in regulating red cell turnover. Further characterization of this pathway may also identify new targets for the treatment of P. falciparum malaria. © 2005 Blackwell Publishing Ltd

Proteomic and Biochemical profiling of 4.1R deficient red blood cells

Eryptosis is a term used to describe the highly regulated homeostatic process of erythrocyte programmed cell death. It mimics apoptosis in nucleated cells in that cell shrinkage and Phosphatidylserine (PS) exposure at the outer membrane leaflet are observed. Recently our group reported that the ligation of a specific synthetic peptide (4N1K), a natural ligand (thrombospondin-1) and a monoclonal antibody (BRIC- 126) which all bind to red cell CD47 glycoprotein (also known as Integrin associated protein) can trigger PS exposure in erythrocytes. We have also demonstrated that several key red cell membrane proteins 4.1R/p55/Glycophorin C (GPC) are involved in the CD47 pathway. This is primarily because we have demonstrated a direct protein-proteininteraction between CD47 and p4.1 and p55. In order to explore these interactions in more depth, we have studied red cells obtained from 4.1R deficient individuals using a combination of differential proteomic profiling and biochemical analysis of the red cell PS-exposure pathways. Interestingly GPC has a interaction with p55 and p4.1Rwhich has been well established. We have used proteomic profiling, use of CD47 ligands to explore PS exposure pathways, protein immunoblotting and flow cytometry on 4.1R deficient red cells in these studies. Our findings have produced significant differences compared with that of normal control age-matched erythrocytes. We show that4.1R deficient RBCs lack, or are significantly deficient in several membrane and membrane skeletal components, and give novel insight into the complexes that 4.1R maintains in the mature RBC. This may suggest it plays a key role in eryptosis, especially pertinent as it is known to have binding sites for PS. Protein immunoblotting showed absence of protein 4.1R and protein p55 confirming earlier studies. Glycophorin C was also diminished and Glycophorin A appeared equal as previously reported. However, previously unreported results included a deficiency of CD44, alterations in CD47 expression, lack of GPC dimerisation, and deficiency of aldolase A. Further we showed that PS exposure is increased in the CD47 pathway (when challenged with CD47 ligands BRIC 126 and 4N1K) in cells lacking protein 4.1R compared with control cells, but decreased in the GPC pathway (when challenged with BRIC 10) in cells lacking protein 4.1R compared with control cells. 4.1R(Madrid) cells were also shown to be resistant to TSP-1 mediated eryptosis. This difference in PS exposure suggests an important regulatory role for 4.1R in both of these apoptotic pathways, and may suggest that CD47-induced PS exposure occurs independently of binding to 4.1R/p55.Studies are ongoing with red cells obtained from a further 4.1R deficient individual (4.1R-/- Lille) and initial analysis is confirms the alterations seen in the 4.R-/- Madrid sample

Noninvasive prenatal diagnosis of fetal Rhesus D: ready for Prime(r) Time

Rhesus (Rh) D blood group incompatibility between the pregnant woman and her fetus is a significant problem due to the possibility of maternal alloimmunization and consequent hemolytic disease of the newborn. The RhD-negative blood group is found in 15% of whites, 3-5% of black Africans, and is rare in Asians. Advances in both our understanding of the RHD locus and its variants, as well as technical improvements in the extraction and amplification of cell-free fetal DNA in maternal plasma, have led to incorporation of noninvasive diagnosis of RHD genotype into routine prenatal care in the United Kingdom, France, and the Netherlands. In this commentary we examine the experience to date with large-scale clinical trials performed in the European Union, describe approaches to reduce false-positive and false-negative results, and review ongoing research to standardize assays and reduce costs using automated assays. False-negative cases are mainly due to either a lack of fetal DNA in the maternal sample due to early gestation or insensitive methods. False-positive cases are due to genotypic variants observed in individuals of African ancestry. Noninvasive prenatal diagnosis of fetal Rhesus D genotype is sensitive and accurate and has been widely validated in Europe. The United States should begin to undertake clinical trials to bring this technology to patient care as soon as possibl

Post-genomics studies and their application to non-invasive prenatal diagnosis

Non-invasive prenatal diagnosis (NIPD) offers the opportunity to eliminate completely the risky procedures of amniocentesis and chorionic villus sampling. The development of NIPD tests has largely centred around the isolation and analysis of fetal cells in the maternal circulation and the analysis of free fetal DNA in maternal plasma. Both of these techniques offer difficult technical challenges, and at the current moment in time the use of free fetal DNA is the simplest and most effective method of defining paternally inherited fetal genes for diagnosis. Post-genomics technologies that explore the proteins (proteomics) and transcripts (transcriptomics) released by the placenta into the maternal circulation offer new opportunities to identify genes and their protein products that are key diagnostic markers of disease (in particular Down syndrome), and might replace the current screening markers in use for prediction of risk of Down syndrome. In the ideal situation, these markers are sufficiently diagnostic not to require invasive sampling of fetal genetic material. Post-genomics techniques might also offer better opportunities for defining fetal cell-specific markers that might enhance their isolation from maternal blood samples. This review describes progress in these studies, particularly those funded by the Special Non-invasive Advances in Fetal and Neonatal Evaluation (SAFE) Network of Excellence. © 2008