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Jean-pierre Cartron - One of the best experts on this subject based on the ideXlab platform.
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Immunoblot analysis of hRHAG and RhD expression in transgenic mice.
2013Co-Authors: Dominique Goossens, Isabelle Mouro-chanteloup, Nelly Da Silva, Sylvain Metral, Ulrich Cortes, Isabelle Callebaut, Julien Picot, Jean-pierre CartronAbstract:Immunoblot from red cell ghost preparations immunostained with anti-hRHAG (LA18.18), anti-RhD (LOR15C9), and anti-actin as loading control. Lane 1= RHD single transgenic TG_RHD-BAC1, lane 2 = double transgenic cross of TG_hRHAG_68.08 with RHD-BAC1, lane 3 = double transgenic cross of TG_hRHAG_68.08 with TG_ RHD_65.08, lane 4 hRHAG single transgenic TG_hRHAG_68.08, lane 5=WT and lane 6= human RhD-positive control. hRHAG protein is seen in all mice with the hRHAG transgene, whereas RhD expression is detected only in the double transgenic mouse carrying both human RHAG and RHD_65.08 transgenes.
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Mice Expressing RHAG and RHD Human Blood Group Genes
PLoS ONE, 2013Co-Authors: Dominique Goossens, Isabelle Mouro-chanteloup, Nelly Da Silva, Sylvain Metral, Ulrich Cortes, Isabelle Callebaut, Julien Picot, Jean-pierre CartronAbstract:Anti-RhD prophylaxis of haemolytic disease of the fetus and newborn (HDFN) is highly effective, but as the suppressive mechanism remains uncertain, a mouse model would be of interest. Here we have generated transgenic mice expressing human RHAG and RhD erythrocyte membrane proteins in the presence and, for human RHAG, in the absence, of mouse RHAG. Human RHAG associates with mouse Rh but not mouse RHAG on red blood cells. In RHAG knockout mice transgenic for human RHAG, the mouse Rh protein is " rescued " (re-expressed), and co-immunoprecipitates with human RHAG, indicating the presence of hetero-complexes which associate mouse and human proteins. RhD antigen was expressed from a human RHD gene on a BAC or from RHD cDNA under control of β-globin regulatory elements. RhD was never observed alone, strongly indicative that its expression absolutely depends on the presence of transgenic human RHAG. This first expression of RhD in mice is an important step in the creation of a mouse model of RhD allo-immunisation and HDFN, in conjunction with the Rh-RHAG knockout mice we have developed previously.
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Generation and characterisation of Rhd and RHAG null mice
British journal of haematology, 2009Co-Authors: Dominique Goossens, Yves Colin, Marie-marcelle Trinh-trang-tan, Martine Debbia, Pierre Ripoche, Camilo Vilela-lamego, Fawzia Louache, William Vainchenker, Jean-pierre CartronAbstract:Summary Mouse Rhd* and RHAG* genes were targeted using insertional vectors; the resulting knockout mice, and double-knockout descendants, were analysed. RHAG glycoprotein deficiency entailed defective assembly of the erythroid Rh complex with complete loss of Rh and intercellular adhesion molecule 4 (ICAM-4), but not CD47, expression. Absence of the Rh protein induced a loss of ICAM-4, and only a moderate reduction of RHAG expression. Double knockout phenotype was similar to that of RHAG targeted mice. Rhd and RHAG deficient mice exhibited neither the equivalent of human Rhnull haemolytic anaemia nor any clinical or cellular abnormalities. Rhd−/− and RHAG−/− erythrocytes showed decreased basal adhesion to an endothelial cell line resulting from defective ICAM-4 membrane expression. There was no difference in recovery from phenylhydrazine-induced haematopoietic stress for double knockout mice as compared to controls, suggesting that ICAM-4 might be dispensable during stress erythropoiesis. Ammonia and methylammonia transport in erythrocytes was severely impaired in RHAG−/− but only slightly in Rhd−/− animals that significantly expressed RHAG, supporting the view that RHAG and RHAG, but not Rh, may act as ammonium transporters in human and mouse erythrocytes. These knockout mice should prove useful for further dissecting the physiological roles of Rh and RHAG proteins in the red cell membrane.
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Generation of mice with inactivated Rh or RHAG genes.
Transfusion clinique et biologique : journal de la Societe francaise de transfusion sanguine, 2006Co-Authors: D Goossens, Pierre Gane, Y Colin, V Bony, Jean-pierre CartronAbstract:Mice carrying inactivated Rh and RHAG genes were generated by insertional targeting. KO animals exhibited normal growth, development and fertility and both types were indistinguishable at a gross phenotypic level from their wild type littermates. Preliminary analysis revealed that red cells from Rh-/- mice lack Rh protein and have a moderate decrease of RHAG protein, whereas those from RHAG-/- mice have a total absence of RHAG and Rh proteins. Studies are in progress to delineate the antigenic, biochemical and functional abnormalities of red cells from these animals as well as the impact on hematological parameters and erythropoiesis.
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Role of RHAG and AQP1 in NH3 and CO2 gas transport in red cell ghosts: a stopped-flow analysis.
Transfusion clinique et biologique : journal de la Societe francaise de transfusion sanguine, 2006Co-Authors: P Ripoche, Pierre Gane, D Goossens, O Devuyst, Y Colin, A S Verkman, Jean-pierre CartronAbstract:To clarify the potential role Rh/RHAG and AQP1 proteins in erythrocyte gas transport, NH3 and CO2 transport was measured in erythrocyte ghost membrane vesicles from rare human variants (Rh(null), CO(null),) and knockout mice (homozygous AQP1-/-, Rh-/- and RHAG-/-) exhibiting well-characterized protein defects. Transport was measured from intracellular pH (pHi) changes in a stopped-flow fluorimeter. NH3 transport was measured in chloride-free conditions with ghosts exposed to 20 mM inwardly directed gradients of gluconate salts of ammonium, hydrazine and methylammonium at 15 degrees C. Alkalinization rates of control samples were 6.5+/-0.3, 4.03+/-0.17, 0.95+/-0.08 s(-1) for each solute, respectively, but were significantly reduced for Rh(null) and CO(null) samples that are deficient in RHAG and AQP1 proteins, respectively. Alkalinization rates of Rh(null) ghosts were about 60%, 83% and 94% lower than that in control ghosts, respectively, for each solute. In CO(null) ghosts, the lack of AQP1 resulted in about 30% reduction of the alkalinization rates as compared to controls, but the transport selectivity of RHAG for the three solutes was preserved. Similar observations were made with ghosts from KO mice RHAG-/- and AQP1-/-. These results confirm the major contribution of RHAG/RHAG in the NH3 conductance of erythrocytes and suggest that the reduction of transport rates in the absence of AQP1 would be better explained by a direct or indirect effect on RHAG/RHAG-mediated transport. When ghosts were preloaded with carbonic anhydrase and exposed to a 25 mM CO2/HCO3- gradient at 6 degrees C, an extremely rapid kinetics of acidification corresponding to CO2 influx was observed. The rate constants were not significantly different between controls and human variants (125+/-6 s(-1)), or between wild-type and KO mice, suggesting no major role of RHAG or AQP1 in CO2 transport, at least in our experimental conditions.
Yves Colin - One of the best experts on this subject based on the ideXlab platform.
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Human RHAG ammonia channel is impaired by the Phe65Ser mutation in overhydrated stomatocytic red cells
American journal of physiology. Cell physiology, 2011Co-Authors: Sandrine Genetet, Yves Colin, Jean Delaunay, Julien Picot, Pierre Ripoche, Sylvain Bigot, Corinne Armari-alla, Isabelle Mouro-chanteloupAbstract:In red cells, Rh-associated glycoprotein (RHAG) acts as an ammonia channel, as demonstrated by stopped-flow analysis of ghost intracellular pH (pHi) changes. Recently, overhydrated hereditary stoma...
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F65S Mutation in RHAG is Associated with Decreased Ammonia Flux Through Overhydrated Stomatocytic Erythrocytes
Biophysical Journal, 2011Co-Authors: Sandrine Genetet, Yves Colin, Jean Delaunay, Julien Picot, Pierre Ripoche, Sylvain Bigot, Corinne Amari-alla, Isabelle Mouro-chanteloupAbstract:The Overhydrated Stomatocytic (OHSt) erythrocytes of four patients, characterized by the F65S mutation (Bruce et al, Blood 2009) located in the pore of the ammonia channel Rh Associated Glycoprotein (RHAG), were studied. We have previously demonstrated that the equivalent substitution (F74L) in the non-erythroid analogue RhCG resulted in a reduction of the ammonia influx of 50% in transfected HEK293 cells.Ghosts prepared by hypotonic lysis of OHSt and control (Ctl) erythrocytes, resealed in the presence of a pH-sensitive probe (pyranine), exhibited echinocytic morphology. Images of 1667 (Ctl) and 1998 (OHSt) echinocytes, visualized by light microscopy, allowed the determination of average ghost diameters: 5.81±0.2 µm (Ctl) and 5.83±0.06 µm (OHSt). RHAG densities, as determined by flow cytometry, were similar for Ctl and OHSt (78 000 to 80 000 copies/cell).Ammonium permeability (P’NH3) was measured by following the internal compartment alkalinisation, after submitting ghosts to inward ammonium gradients. Alkalinisation rate constants (k) were deduced from stopped-flow analysis of pH variations at 15°C, allowing the determination of P’NH3 by using the simplified equation P’NH3=k.r/3 (r: radius of spheric ghosts). The P’NH3 values were significantly different between Ctl and OHSt samples (1.7±0.11 vs 0.71±0.004 µm.s−1).Additionally, water permeability was measured in the same OHSt resealed ghosts but containing 8mM 6-carboxyfluorescein (6-CF), after submitting them to an osmotic gradient (150mosm/kg/H2O mannitol). The osmotic water permeability (Pf) values deduced from the fluorescence quenching at 15°C were similar between Ctl and OHSt samples (0.035±0.005 vs 0.035±0.04 cm.s−1).In conclusion, the F65S mutation of RHAG induces a reduction of ammonia flux resulting in an alteration of pHi regulation. The decrease of ammonia conductance through the mutated RHAG can result from loss of hydrophobicity and/or from structural modifications inside the pore of the channel.
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Generation and characterisation of Rhd and RHAG null mice
British journal of haematology, 2009Co-Authors: Dominique Goossens, Yves Colin, Marie-marcelle Trinh-trang-tan, Martine Debbia, Pierre Ripoche, Camilo Vilela-lamego, Fawzia Louache, William Vainchenker, Jean-pierre CartronAbstract:Summary Mouse Rhd* and RHAG* genes were targeted using insertional vectors; the resulting knockout mice, and double-knockout descendants, were analysed. RHAG glycoprotein deficiency entailed defective assembly of the erythroid Rh complex with complete loss of Rh and intercellular adhesion molecule 4 (ICAM-4), but not CD47, expression. Absence of the Rh protein induced a loss of ICAM-4, and only a moderate reduction of RHAG expression. Double knockout phenotype was similar to that of RHAG targeted mice. Rhd and RHAG deficient mice exhibited neither the equivalent of human Rhnull haemolytic anaemia nor any clinical or cellular abnormalities. Rhd−/− and RHAG−/− erythrocytes showed decreased basal adhesion to an endothelial cell line resulting from defective ICAM-4 membrane expression. There was no difference in recovery from phenylhydrazine-induced haematopoietic stress for double knockout mice as compared to controls, suggesting that ICAM-4 might be dispensable during stress erythropoiesis. Ammonia and methylammonia transport in erythrocytes was severely impaired in RHAG−/− but only slightly in Rhd−/− animals that significantly expressed RHAG, supporting the view that RHAG and RHAG, but not Rh, may act as ammonium transporters in human and mouse erythrocytes. These knockout mice should prove useful for further dissecting the physiological roles of Rh and RHAG proteins in the red cell membrane.
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Functional interaction between Rh proteins and the spectrin-based skeleton in erythroid and epithelial cells.
Transfusion Clinique Et Biologique, 2006Co-Authors: Virginie Nicolas, Jean-pierre Cartron, Pierre Gane, Caroline Le Van Kim, Isabelle Mouro-chanteloup, Claude Lopez, A. Gimm, Narla Mohandas, Yves ColinAbstract:Abstract We summarize the different experimental approaches which provide evidence that direct interaction of Rh and RHAG to ankyrin-R constitutes, together with the AE-1 (Band 3)-ankyrin-protein 4.2 and GPC-protein 4.1-p55 complexes, another major anchoring site between the red cell membrane bilayer and the underlying spectrin-based skeleton. The observations that some residues of the ankyrin binding site are mutated in Rh and RHAG proteins from some weak D and Rh null variants, respectively, suggest that the Rh-RHAG/ankyrin-R interaction plays a crucial role in the biosynthesis and/or the stability of the Rh complex in the red cell membrane. Similarly, binding to ankyrin G is required for cell surface expression of the non-erythroid member of the Rh protein family, RhBG, at the basolateral membrane domain of polarized epithelial cells. The next challenge will be to determine whether binding to the membrane skeleton may be critical for the emerging ammonium transport function of Rh proteins in erythroid and non-erythroid cells.
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Rh proteins: key structural and functional components of the red cell membrane.
Blood reviews, 2005Co-Authors: Caroline Le Van Kim, Yves Colin, Jean-pierre CartronAbstract:Rh (Rhesus) proteins (D, CcEe) are expressed in red cells (RBC) in association with other membrane proteins (RHAG, LW, CD47 and GPB). By interacting with the spectrin-based skeleton through protein 4.2 and ankyrin, the Rh complex contributes to the maintenance of the mechanical properties of the erythrocyte membrane. The RH system is one of the most immunogenic and polymorphic human blood group system. Molecular basis of most Rh phenotypes, including the Rh(null) phenotype associated with hemolytic anemia, have been determined. The demonstration that the RHD-positive locus is composed of the RHD and RHCE genes, whereas the RHD gene is deleted in most RhD-negative individuals, allowed fetal RhD genotyping by non-invasive PCR assays for antenatal diagnosis of pregnancy at risk for Rh hemolytic disease of the newborn. In mammals, the Rh protein family includes two non-erythroid members, RhBG and RhCG, mainly expressed in liver and kidney, two organs specialized in ammonia genesis and excretion. Functional analyses in heterologous systems revealed that RHAG, RhBG and RhCG can mediate ammonium (NH(3) and/or NH(4)(+)) transport across the cell membrane and might represent mammalian specific ammonium transporters. Furthermore, recent studies performed in human and murine red blood cells (RBC) indicate that RHAG facilitates CH(3)NH(2)/NH(3) movement across the membrane and represents a potential example of gas channel. The crystallographic structure of the bacterial ammonia channel AmtB and functional studies showing that AmtB conducts NH(3) into reconstituted vesicles is fully consistent with these latter studies. In RBCs, RHAG may transport NH(3) to detoxifying organs like kidney and liver and with non-erythroid tissues orthologs may contribute to regulation of the acid-base balance.
Isabelle Mouro-chanteloup - One of the best experts on this subject based on the ideXlab platform.
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Immunoblot analysis of hRHAG and RhD expression in transgenic mice.
2013Co-Authors: Dominique Goossens, Isabelle Mouro-chanteloup, Nelly Da Silva, Sylvain Metral, Ulrich Cortes, Isabelle Callebaut, Julien Picot, Jean-pierre CartronAbstract:Immunoblot from red cell ghost preparations immunostained with anti-hRHAG (LA18.18), anti-RhD (LOR15C9), and anti-actin as loading control. Lane 1= RHD single transgenic TG_RHD-BAC1, lane 2 = double transgenic cross of TG_hRHAG_68.08 with RHD-BAC1, lane 3 = double transgenic cross of TG_hRHAG_68.08 with TG_ RHD_65.08, lane 4 hRHAG single transgenic TG_hRHAG_68.08, lane 5=WT and lane 6= human RhD-positive control. hRHAG protein is seen in all mice with the hRHAG transgene, whereas RhD expression is detected only in the double transgenic mouse carrying both human RHAG and RHD_65.08 transgenes.
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Mice Expressing RHAG and RHD Human Blood Group Genes
PLoS ONE, 2013Co-Authors: Dominique Goossens, Isabelle Mouro-chanteloup, Nelly Da Silva, Sylvain Metral, Ulrich Cortes, Isabelle Callebaut, Julien Picot, Jean-pierre CartronAbstract:Anti-RhD prophylaxis of haemolytic disease of the fetus and newborn (HDFN) is highly effective, but as the suppressive mechanism remains uncertain, a mouse model would be of interest. Here we have generated transgenic mice expressing human RHAG and RhD erythrocyte membrane proteins in the presence and, for human RHAG, in the absence, of mouse RHAG. Human RHAG associates with mouse Rh but not mouse RHAG on red blood cells. In RHAG knockout mice transgenic for human RHAG, the mouse Rh protein is " rescued " (re-expressed), and co-immunoprecipitates with human RHAG, indicating the presence of hetero-complexes which associate mouse and human proteins. RhD antigen was expressed from a human RHD gene on a BAC or from RHD cDNA under control of β-globin regulatory elements. RhD was never observed alone, strongly indicative that its expression absolutely depends on the presence of transgenic human RHAG. This first expression of RhD in mice is an important step in the creation of a mouse model of RhD allo-immunisation and HDFN, in conjunction with the Rh-RHAG knockout mice we have developed previously.
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Human RHAG ammonia channel is impaired by the Phe65Ser mutation in overhydrated stomatocytic red cells
American journal of physiology. Cell physiology, 2011Co-Authors: Sandrine Genetet, Yves Colin, Jean Delaunay, Julien Picot, Pierre Ripoche, Sylvain Bigot, Corinne Armari-alla, Isabelle Mouro-chanteloupAbstract:In red cells, Rh-associated glycoprotein (RHAG) acts as an ammonia channel, as demonstrated by stopped-flow analysis of ghost intracellular pH (pHi) changes. Recently, overhydrated hereditary stoma...
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F65S Mutation in RHAG is Associated with Decreased Ammonia Flux Through Overhydrated Stomatocytic Erythrocytes
Biophysical Journal, 2011Co-Authors: Sandrine Genetet, Yves Colin, Jean Delaunay, Julien Picot, Pierre Ripoche, Sylvain Bigot, Corinne Amari-alla, Isabelle Mouro-chanteloupAbstract:The Overhydrated Stomatocytic (OHSt) erythrocytes of four patients, characterized by the F65S mutation (Bruce et al, Blood 2009) located in the pore of the ammonia channel Rh Associated Glycoprotein (RHAG), were studied. We have previously demonstrated that the equivalent substitution (F74L) in the non-erythroid analogue RhCG resulted in a reduction of the ammonia influx of 50% in transfected HEK293 cells.Ghosts prepared by hypotonic lysis of OHSt and control (Ctl) erythrocytes, resealed in the presence of a pH-sensitive probe (pyranine), exhibited echinocytic morphology. Images of 1667 (Ctl) and 1998 (OHSt) echinocytes, visualized by light microscopy, allowed the determination of average ghost diameters: 5.81±0.2 µm (Ctl) and 5.83±0.06 µm (OHSt). RHAG densities, as determined by flow cytometry, were similar for Ctl and OHSt (78 000 to 80 000 copies/cell).Ammonium permeability (P’NH3) was measured by following the internal compartment alkalinisation, after submitting ghosts to inward ammonium gradients. Alkalinisation rate constants (k) were deduced from stopped-flow analysis of pH variations at 15°C, allowing the determination of P’NH3 by using the simplified equation P’NH3=k.r/3 (r: radius of spheric ghosts). The P’NH3 values were significantly different between Ctl and OHSt samples (1.7±0.11 vs 0.71±0.004 µm.s−1).Additionally, water permeability was measured in the same OHSt resealed ghosts but containing 8mM 6-carboxyfluorescein (6-CF), after submitting them to an osmotic gradient (150mosm/kg/H2O mannitol). The osmotic water permeability (Pf) values deduced from the fluorescence quenching at 15°C were similar between Ctl and OHSt samples (0.035±0.005 vs 0.035±0.04 cm.s−1).In conclusion, the F65S mutation of RHAG induces a reduction of ammonia flux resulting in an alteration of pHi regulation. The decrease of ammonia conductance through the mutated RHAG can result from loss of hydrophobicity and/or from structural modifications inside the pore of the channel.
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Functional interaction between Rh proteins and the spectrin-based skeleton in erythroid and epithelial cells.
Transfusion Clinique Et Biologique, 2006Co-Authors: Virginie Nicolas, Jean-pierre Cartron, Pierre Gane, Caroline Le Van Kim, Isabelle Mouro-chanteloup, Claude Lopez, A. Gimm, Narla Mohandas, Yves ColinAbstract:Abstract We summarize the different experimental approaches which provide evidence that direct interaction of Rh and RHAG to ankyrin-R constitutes, together with the AE-1 (Band 3)-ankyrin-protein 4.2 and GPC-protein 4.1-p55 complexes, another major anchoring site between the red cell membrane bilayer and the underlying spectrin-based skeleton. The observations that some residues of the ankyrin binding site are mutated in Rh and RHAG proteins from some weak D and Rh null variants, respectively, suggest that the Rh-RHAG/ankyrin-R interaction plays a crucial role in the biosynthesis and/or the stability of the Rh complex in the red cell membrane. Similarly, binding to ankyrin G is required for cell surface expression of the non-erythroid member of the Rh protein family, RhBG, at the basolateral membrane domain of polarized epithelial cells. The next challenge will be to determine whether binding to the membrane skeleton may be critical for the emerging ammonium transport function of Rh proteins in erythroid and non-erythroid cells.
Cheng-han Huang - One of the best experts on this subject based on the ideXlab platform.
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Sequence, Organization, and Evolution of Rh50 Glycoprotein Genes in Nonhuman Primates
Journal of molecular evolution, 2000Co-Authors: Cheng-han Huang, P. A. Apoil, Zhi Liu, Antoine BlancherAbstract:The human RHAG locus encodes Rh50 glycoprotein, a polytopic protein that modulates expression of Rh antigens carried by Rh30 polypeptides. Rh50 is almost invariant, whereas Rh30 shows high polymorphism. To assess the relative conservation and phylogenetic relationship of RHAG genes, we characterized their protein expression, transcript structure, genomic organization, and noncoding regions (promoter and introns) in seven nonhuman primate species. Western blot showed that only ape Rh50 glycoproteins are recognized by the antibody 2D10 specific for the human counterpart. Analysis of RHAG gene and its transcript showed a high degree of sequence identity and features of interspecific diversity. The nonhuman primate RHAG genes are highly similar in promoter region and identical in exon-intron organization. Genomic sequencing identified one retro-transposon-like element in intron 2 and three types of Alu elements in intron 4 and 9, with varying copies of minisatellites. Reconstruction of coding and noncoding sequence trees revealed concordances and discordances with regard to the branching of RHAG-like genes in higher primates. A joined tree of Rh50 glycoproteins and Rh30 polypeptides shows that the former evolved at a rate about two times slower than the latter. Statistical tests demonstrated that at least a portion of the RHAG gene was subjected to a positive selection during evolution of anthropoids.
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The Mouse Rhl1 and RHAG Genes: Sequence, Organization, Expression, and Chromosomal Mapping
Biochemical Genetics, 1999Co-Authors: Zhi Liu, Cheng-han HuangAbstract:To seek an alternative model for studies of theRh protein complex, we isolated by homology cloning andcharacterized the mouse Rhced and RHAG genes, which are homologous to the human RH and RHAG genes,respectively. Rhced encodes a glycoprotein of418 amino acids which occurs as a composite of human RhDand RhCE with 60% identity and 74% similarity. RHAGencodes a glycoprotein of 438 amino acids thatshares 79% identity and 87% similarity to humanRh50. However, RHAG has an elongated C terminus and fourN-glycosylation sites clustered on exoloop 1. Hydropathyplots suggest that Rhl1 and RHAG each spanthe lipid bilayer 12 times, with N and Ctermini facing the cytoplasm. Rhced and RHAG are bothspecified by 10 exons and bear a similar exon/intronstructure, but their major transcription start sites aremapped at –17A and –27A. Northernanalysis revealed coexpression of Rhced and RHAG from11-day embryos throughout adult life in erythroidtissues. Southern blotting and linkage analysis showedthat Rhced and RHAG are single-copygenes localized to chromosomes 4 and 17, respectively;they are paralogous to one another but orthologous tohuman RH and RHAG . The resultstogether predate the occurrence and signifya conserved function of the erythroid-specificRh membrane structures.
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Molecular basis for Rhnull syndrome: Identification of three new missense mutations in the Rh50 glycoprotein gene
American journal of hematology, 1999Co-Authors: Cheng-han Huang, Zhi Liu, Marion E. Reid, Guangjie Cheng, Ying Chen, Gregory R. Halverson, Yasuto OkuboAbstract:Rh(null) is a rare autosomal recessive disorder characterized by an absence of Rh antigens and a varying degree of hemolytic anemia and spherostomatocytosis. We report studies of two Japanese Rh(null) cases and describe three new missense mutations of RHAG, the locus that encodes Rh50 glycoprotein and modulates Rh antigen expression. In Rh(null)(HT), RHAG harbored in exon 6 two G-->A transitions, GTT-->ATT and GGA-->AGA, which cause Val(270)-->Ile and Gly(280)-->Arg substitutions, respectively. These missense mutations were cotransmitted from the propositus to the children and were predicted to reside in endoloop 5 and transmembrane (TM) segment 9, respectively. In Rh(null)(WO), RHAG contained in exon 9 a single G-->T transversion, GGT-->GTT, which caused a Gly(380)-->Val missense change in TM12 segment. The G-->T transversion, which is located at the +1 position of exon 9, had also affected pre-mRNA splicing and caused partial exon skipping. Although both Rh(null) cases had a structurally normal RH antigen locus, hemagglutination and immunoblotting showed no expression of Rh antigens or proteins. These results correlate each mutation with a structural defect in the respective TM domain of Rh50 glycoprotein.
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Rhmod syndrome: a family study of the translation-initiator mutation in the Rh50 glycoprotein gene.
American journal of human genetics, 1999Co-Authors: Cheng-han Huang, G.-j. Cheng, Y. ChenAbstract:Rhmod syndrome is a rare genetic disorder thought to result from mutations at a "modifier" but not at the suppressor underlying the regulator type of Rhnull disease. We studied this disorder in a Jewish family with a consanguineous background and analyzed RH and RHAG, the two loci that control Rh-antigen expression and Rh-complex assembly. Despite the presence of a d (D-negative) haplotype, no other gross alteration was found at RH, and cDNA sequencing showed a normal structure for D, Ce, and ce Rh transcripts in family members. However, analysis of RHAG transcript, which encodes Rh50 glycoprotein, identified a single G-->T transversion in the initiation codon, causing a missense amino acid change (ATG[Met]-->ATT[Ile]). This point mutation also occurred in the genomic region spanning exon 1 of RHAG, and its genotypic status in the mother and two children was confirmed by analysis of single-strand conformation polymorphism. Although blood typing showed a very weak expression of Rh antigens, immunoblotting barely detected the Rh proteins in the Rhmod membrane. In vitro transcription-coupled translation assays showed that the initiator mutants of Rhmod-but not those of the wild type-could be translated from ATG codons downstream. Our findings point to incomplete penetrance of the Rhmod mutation, in the form of "leaky" translation, leading to some posttranslational defects affecting the structure, interaction, and processing of Rh50 glycoprotein.
Emile Van Den Akker - One of the best experts on this subject based on the ideXlab platform.
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a variant RHAG protein encoded by the RHAG 572a allele causes serological weak d expression while maintaining normal rhce phenotypes
Transfusion, 2019Co-Authors: Jizhi Wen, O. J. H. M. Verhagen, Shuangshuang Jia, Qian-ni Liang, Zhen Wang, Ling Wei, Hong Luo, Guangping Luo, Gestur Vidarsson, Emile Van Den AkkerAbstract:BACKGROUND: The molecular events resulting in a weak D phenotype include missense mutations, in-frame insertion, or deletion mutations of the RHD gene and hybrid RHD-CE-D hybrid alleles. Mutations in genes encoding the proteins that are required for proper membrane expression of Rh proteins, such as RHAG and ankyrin 1, can lead to absent or weakened expression of Rh antigens. STUDY DESIGN AND METHODS: Blood sample from a Chinese blood donor with a serological weak D phenotype was collected. RHAG antigen expression, RhD, and RhCE phenotypes were determined. Analysis of the RHD and RHCE genotypes by RH multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing of the RHD exons, and next-generation sequencing (NGS) of the RHAG and ANK1 exons were performed. Expression studies in vitro were conducted by lentivirally transducing the mutant RHAG*572A or wild-type RHAG, in combination with either RHD or RHCE constructs, into HEK 293 T cells. The expression of RHAG, RhD, and RhCE antigens was analyzed by flow cytometry. RESULTS: Serological weak D and normal C + c-E–e + phenotypes, normal CCDDee genotype determined by RH-MLPA, and normal sequence of the RHD gene by Sanger sequencing were demonstrated. A homozygous variant (c.572G > A, p.Arg191Gln) of the RHAG gene was revealed by NGS analysis. Normal RHAG, weak RhD, and normal RhCE antigens were detected in cells transduced with the mutant RHAG*572A, the mutant RHAG*572A and RHD, and the mutant RHAG*572A and RHCE constructs, respectively. CONCLUSION: The homozygous presence of RHAG*572A allele results in weak D expression. It does not affect RhCE expression.
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A variant RHAG protein encoded by the RHAG*572A allele causes serological weak D expression while maintaining normal RhCE phenotypes.
Transfusion, 2018Co-Authors: Jizhi Wen, O. J. H. M. Verhagen, Shuangshuang Jia, Qian-ni Liang, Zhen Wang, Ling Wei, Hong Luo, Guangping Luo, Gestur Vidarsson, Emile Van Den AkkerAbstract:BACKGROUND: The molecular events resulting in a weak D phenotype include missense mutations, in-frame insertion, or deletion mutations of the RHD gene and hybrid RHD-CE-D hybrid alleles. Mutations in genes encoding the proteins that are required for proper membrane expression of Rh proteins, such as RHAG and ankyrin 1, can lead to absent or weakened expression of Rh antigens. STUDY DESIGN AND METHODS: Blood sample from a Chinese blood donor with a serological weak D phenotype was collected. RHAG antigen expression, RhD, and RhCE phenotypes were determined. Analysis of the RHD and RHCE genotypes by RH multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing of the RHD exons, and next-generation sequencing (NGS) of the RHAG and ANK1 exons were performed. Expression studies in vitro were conducted by lentivirally transducing the mutant RHAG*572A or wild-type RHAG, in combination with either RHD or RHCE constructs, into HEK 293 T cells. The expression of RHAG, RhD, and RhCE antigens was analyzed by flow cytometry. RESULTS: Serological weak D and normal C + c-E–e + phenotypes, normal CCDDee genotype determined by RH-MLPA, and normal sequence of the RHD gene by Sanger sequencing were demonstrated. A homozygous variant (c.572G > A, p.Arg191Gln) of the RHAG gene was revealed by NGS analysis. Normal RHAG, weak RhD, and normal RhCE antigens were detected in cells transduced with the mutant RHAG*572A, the mutant RHAG*572A and RHD, and the mutant RHAG*572A and RHCE constructs, respectively. CONCLUSION: The homozygous presence of RHAG*572A allele results in weak D expression. It does not affect RhCE expression.
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Critical band 3 multiprotein complex interactions establish early during human erythropoiesis
Blood, 2011Co-Authors: Timothy J. Satchwell, Emile Van Den Akker, Geoff Daniels, David J. Anstee, Amanda J. Bell, Stephanie Pellegrin, Sabine Kupzig, Kay Ridgwell, Ashley M. ToyeAbstract:Band 3, the major anion transport protein of human erythrocytes, forms the core of a multiprotein complex in the erythrocyte membrane. Here we studied the spatiotemporal mechanisms of band 3 multiprotein complex assembly during erythropoiesis. Significant pools of intracellular band 3 and Rh-associated glycoprotein (RHAG) were found in the basophilic erythroblast. These intracellular pools decreased in the polychromatic erythroblast, whereas surface expression increased and were lowest in the orthochromatic erythroblast and reticulocytes. Protease treatment of intact cells to remove extracellular epitopes recognized by antibodies to band 3 and RHAG was used to study surface delivery kinetics and intracellular complex composition from the proerythroblast stage to the enucleated reticulocyte. Newly synthesized band 3 and protein 4.2 interact initially in the early stages of the secretory pathway and are found associated at the plasma membrane from the basophilic stage of erythropoiesis. Although we could successfully coimmunoprecipitate Rh with RHAG from plasma membrane pools at a similar stage, no intracellular interaction between these proteins was detectable. Knockdown of RHAG during early erythropoiesis was accompanied by a concomitant drop in membrane expression of Rh polypeptides. These data are consistent with assembly of major components of the band 3 macrocomplex at an early stage during erythropoiesis.
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Mapping the Assembly of Band 3 and Rhesus Multi-Protein Complexes During Erythropoiesis
Blood, 2010Co-Authors: Emile Van Den Akker, Timothy J. Satchwell, Geoff Daniels, Ashley M. ToyeAbstract:Abstract 812 Band 3 forms the core of a large multiprotein complex in the erythrocyte membrane, the Band 3 macrocomplex, which also includes proteins of the Rhesus complex (Rh and RHAG). Mutations in genes encoding proteins within this complex can result in hereditary spherocytosis with varying severity. The effect of distinct mutations and deficiencies in proteins of the Band 3 macrocomplex has been studied in detail in mature erythrocytes. This revealed important functional and structural properties of individual proteins and their relationships with other proteins within the Band 3 macrocomplex. Nevertheless, considerably less is know about the spatio-temporal mechanisms that direct the formation of the Band 3 macrocomplex, and that may explain the aberrations in the complex observed in spherocytosis. Therefore, we studied expression and mutual interactions of proteins of the band3 macrocomplex during development of proerythroblasts to reticulocytes. Using confocal microscopy and western blotting, significant pools of intracellular Band 3 and RHAG were found in the basophilic normoblast. These intracellular pools gradually decreased in the polychromatic normoblast and were absent or low in the orthochromatic normoblast and reticulocytes, while surface expression increased. We used pronase treatment of intact cells to remove extracellular epitopes of BRIC 6 (Band 3 antibody) and LA1818 (RHAG antibody) to study the mechanism by which the intracellular pool of Band 3 and RHAG contributes to formation of the Band 3 complex on the cell surface. Pronase treatment of cells incubated with cycloheximide to block protein synthesis resulted in a reduced but still significant reappearance of BRIC6 (Band 3) and LA1818 (RHAG) epitopes on the plasma membrane confirming the presence of intracellular Band 3 and RHAG pools. It also showed that the bulk of Band 3 and RHAG is synthesized and trafficked to the membrane between the early basophilic and polychromatic stage. Immuneprecipitation of Band 3 from cell lysates of pronase treated cells pre-treated with brefeldin A to collapse the Golgi showed no increase in co-immuneprecipitated protein 4.2 albeit an increase in intracellular Band 3 expression. This suggests that protein 4.2 and Band 3 interact in the first Golgi compartment or late ER. In addition, pre-treatment of cells with cycloheximide prior to pronase treatment resulted in depletion of the intracellular Band 3 and co-immuneprecipitated protein 4.2 pool indicating that Band 3 and protein 4.2 traffic as a complex to the plasma-membrane. We were unable to co-immuneprecipitate Rh or Band 3 with intracellular pools of RHAG, whereas Rh was co-immuneprecipitated with RHAG from the plasma-membrane and from total cell lysates. Knockdown of RHAG in differentiating erythroblasts revealed a concomitant drop in membrane expression of Rh, leaving Band 3 unaffected, indicating that plasma-membrane expression of Rh but not Band 3 is dependent on RHAG. In conclusion, despite the described association between the RHAG complex and the Band 3 complex in erythrocytes, the data suggest that the Band 3-protein 4.2 complex traffics and assembles independently from Rh and RHAG during erythroid differentiation. The experiments suggest that Rh and RHAG do not traffic as a complex to the plasma-membrane but probably assemble in the plasma-membrane. The RHAG knockdown experiments suggest that the dependency of Rh on RHAG as observed in Rhnull syndrome erythrocytes (“Rh regulator type”) originates early during erythropoiesis. Band3 surface expression was not affected upon RHAG knock down, which re-produced the unperturbed Band 3 levels seen in these patients. Disclosures: No relevant conflicts of interest to declare.