Rh Proteins Research Articles

A band 3–based macrocomplex of integral and peripheral proteins in the RBC membrane

AbstractWe have studied the membrane proteins of band 3 anion exchanger (AE1)–deficient mouse and human red blood cells. It has been shown previously that proteins of the band 3 complex are reduced or absent in these cells. In this study we show that proteins of the Rh complex are also greatly reduced (Rh-associated glycoprotein, Rh polypeptides, CD47, glycophorin B) or absent (LW). These observations suggest that the Rh complex is associated with the band 3 complex in healthy RBCs. Mouse band 3(−/−) RBCs differed from the human band 3–deficient RBCs in that they retained CD47. Aquaporin 1 was reduced, and its glycosylation was altered in mouse and human band 3–deficient RBCs. Proteins of the glycophorin C complex, and other proteins with independent cytoskeletal interactions, were present in normal or increased amounts. To obtain direct evidence for the association of the band 3 and the Rh protein complexes in the RBC, we examined whether Rh complex proteins were coimmunoprecipitated with band 3 from membranes. RhAG and Rh were found to be efficiently coimmunoprecipitated with band 3 from deoxycholate-solubilized membranes. Results suggest that band 3 forms the core of a macrocomplex of integral and peripheral RBC membrane proteins. The presence of these proteins in a single structural macrocomplex makes it likely that they have linked functional or regulatory roles. We speculate that this macrocomplex may function as an integrated CO2/O2 gas exchange unit (metabolon) in the erythrocyte.

RhBG and RhCG, the putative ammonia transporters, are expressed in the same cells in the distal nephron.

Two nonerythroid homologs of the blood group Rh proteins, RhCG and RhBG, which share homologies with specific ammonia transporters in primitive organisms and plants, could represent members of a new family of proteins involved in ammonia transport in the mammalian kidney. Consistent with this hypothesis, the expression of RhCG was recently reported at the apical pole of all connecting tubule (CNT) cells as well as in intercalated cells of collecting duct (CD). To assess the localization along the nephron of RhBG, polyclonal antibodies against the Rh type B glycoprotein were generated. In immunoblot experiments, a specific polypeptide of Mr approximately 50 kD was detected in rat kidney cortex and in outer and inner medulla membrane fractions. Immunocytochemical studies revealed RhBG expression in distal nephron segments within the cortical labyrinth, medullary rays, and outer and inner medulla. RhBG expression was restricted to the basolateral membrane of epithelial cells. The same localization was observed in rat and mouse kidney. RT-PCR analysis on microdissected rat nephron segments confirmed that RhBG mRNAs were chiefly expressed in CNT and cortical and outer medullary CD. Double immunostaining with RhCG demonstrated that RhBG and RhCG were coexpressed in the same cells, but with a basolateral and apical localization, respectively. In conclusion, RhBG and RhCG are present in a major site of ammonia secretion in the kidney, i.e., the CNT and CD, in agreement with their putative role in ammonium transport.

Fractional attachment of CD47 (IAP) to the erythrocyte cytoskeleton and visual colocalization with Rh protein complexes

AbstractInteractions of CD47 and RhAG and the Rh proteins are visualized between one another and with the cytoskeleton of intact erythrocytes. In a first study, CD47 is labeled with a phycoerythrin (PhE)– tagged antibody, which generates discrete spots that reflect induced clusters of CD47. Rh and RhAG colocalize with each other and to these induced clusters, whereas Band 3 and glycophorin C remain more homogeneously dispersed on the cell periphery. In a second study, red cells are aspirated into a micropipette, and immunofluorescent maps of the surface gradients that develop for CD47 and RhAG determine cytoskeletal connectivity. CD47 and RhAG gradients on normal red cells prove to be nearly identical and also appear intermediate to those found for the fluid bilayer and network-linked glycophorin C. Similar gradients are obtained for CD47 on Rhnull cells, suggesting that linkage of CD47 to the spectrin-actin skeleton is independent of Rh or RhAG and is not affected by CD47's reduced surface expression on these cells. The results show that CD47 colocalizes with Rh and RhAG but is fractionally attached to the red cell membrane skeleton independent of these and other major integral membrane proteins involved in cytoskeletal attachment. The results imply a homogeneous base distribution of CD47, restrained by cytoskeleton linkages, plus a smaller fraction of CD47, which is able to diffuse in the membrane.

Identification of a unique family of F-box proteins in atadenoviruses.

Ovine adenovirus isolate 287 (OAdV-7) is the prototype of the atadenoviruses, a genus whose strategy for infection and replication is still being elucidated. A transcription unit at the right end of the genome contains four related genes (ORFs RH1, 2, 4, and 6), at least three of which are nonessential for replication in vitro. Related genes are also present in the genomes of bovine and avian atadenoviruses. To investigate how these apparently redundant genes are decoded, a more detailed transcription map of the right end of the OAdV-7 genome has been deduced. Eight transcripts that were derived from a promoter in the terminal repeat sequence were identified. Five were potentially bicistronic. The transcripts could encode all the potential proteins of the region subject to efficient reinitiation of translation. However, the most interesting and surprising finding in this work was that the related RH proteins carry an F-box motif. This was first identified in OAdV-7 RH1 and subsequently found in RH2, 4, and 6 proteins and the related reading frames from the bovine and avian atadenoviruses. Although very rare among viral proteins, several hundred cellular proteins contain F-box motifs. F-box proteins facilitate the degradation of a variety of important regulatory proteins via SCF ubiquitin ligase complexes. Thus, it appears that atadenoviruses have adopted a strategy to regulate a key cellular pathway(s) that distinguishes them from the other adenovirus genera and from most viruses in general.

Rh proteins: a family of structural membrane proteins with putative transport activity.

Rh proteins: a family of structural membrane proteins with putative transport activity.

Expression of RhCG, a new putative NH(3)/NH(4)(+) transporter, along the rat nephron.

Two non-erythroid members of the erythrocyte Rhesus (Rh) protein family, RhBG and RhCG, have been recently cloned in the kidney. These proteins share homologies with specific NH(3)/NH(4)(+) transporters (Mep/Amt) in primitive organisms and plants. When expressed in a Mep-deficient yeast, RhCG can function as a bidirectional NH(3)/NH(4)(+) transporter. The aim of this study was to determine the intrarenal and intracellular location of RhCG in rat kidney. RT-PCR on microdissected rat nephron segments demonstrated expression of mRNAs encoding RhCG in distal convoluted tubules, connecting ducts, and cortical and outer medullary collecting ducts but not in proximal tubules and thick ascending limbs of Henle's loop. Immunolocalization studies performed on rat kidney sections with rabbit anti-human RhCG 31 to 48 antibody showed labeling of the apical pole of tubular cells within the cortex, the outer medulla, and the upper portion of the inner medulla. All cells within connecting tubules had identical apical staining. In cortical collecting ducts, a subpopulation of cells that has either apical staining (alpha-intercalated cells) or diffuse staining (beta-intercalated cells) for the beta1 subunit of the H(+)-ATPase, was heavily stained at their apical pole with the RhCG antibody while principal cells identified as H(+)-ATPase negative cells showed a faint apical staining for RhCG that was much less intense than in adjacent intercalated cells. In the outer medulla and the upper portion of the inner medulla, RhCG labeling was restricted to a subpopulation of cells within the collecting duct that apically express the beta1 subunit of the H(+)-ATPase, indicating that RhCG expression in medullary collecting ducts is restricted to intercalated cells. No labeling was seen in glomeruli, proximal tubules, and limbs of Henle's loop. Immunoblotting of apical membrane fractions from rat kidney cortex with the rabbit anti-human RhCG 31 to 48 antibody revealed a doublet band at approximatively 65 kD.

Complete sequence of Tvv1, a family of Ty 1 copia-like retrotransposons of Vitis vinifera L., reconstituted by chromosome walking.

A chromosome-walking strategy was used to sequence and characterize retrotransposons in the grapevine genome. The reconstitution of a family of retroelements, named Tvv1, was achieved by six successive steps. These elements share a single, highly conserved open reading frame 4,153 nucleotides-long, putatively encoding the gag, pro, int, rt and rh proteins. Comparison of the Tvv1 open reading frame coding potential with those of drosophila copia and tobacco Tnt1, revealed that Tvv1 is closely related to Ty 1 copia-like retrotransposons. A highly variable untranslated leader region, upstream of the open reading frame, allowed us to differentiate Tvv1 variants, which represent a family of at least 28 copies, in varying sizes. This internal region is flanked by two long terminal repeats in direct orientation, sized between 149 and 157 bp. Among elements theoretically sized from 4,970 to 5,550 bp, we describe the full-length sequence of a reference element Tvv1-1, 5,343 nucleotides-long. The full-length sequence of Tvv1-1 compared to pea PDR1 shows a 53.3% identity. In addition, both elements contain long terminal repeats of nearly the same size in which the U5 region could be entirely absent. Therefore, we assume that Tvv1 and PDR1 could constitute a particular class of short LTRs retroelements.

The H2 sensor of Ralstonia eutropha: biochemical and spectroscopic analysis of mutant proteins modified at a conserved glutamine residue close to the [NiFe] active site

[NiFe] hydrogenases contain a highly conserved histidine residue close to the [NiFe] active site which is altered by a glutamine residue in the H(2)-sensing [NiFe] hydrogenases. In this study, we exchanged the respective glutamine residue of the H(2) sensor (RH) of Ralstonia eutropha, Q67 of the RH large subunit HoxC, by histidine, asparagine and glutamate. The replacement by histidine and asparagine resulted in slightly unstable RH proteins which were hardly affected in their regulatory and enzymatic properties. The exchange to glutamate led to a completely unstable RH protein. The purified wild-type RH and the mutant protein with the Gln/His exchange were analysed by continuous-wave and pulsed electron paramagnetic resonance (EPR) techniques. We observed a coupling of a nitrogen nucleus with the [NiFe] active site for the mutant protein which was absent in the spectrum of the wild-type RH. A combination of theoretical calculations with the experimental data provided an explanation for the observed coupling. It is shown that the coupling is due to the formation of a weak hydrogen bond between the protonated N(epsilon) nucleus of the histidine with the sulfur of a conserved cysteine residue which coordinates the metal atoms of the [NiFe] active site as a bridging ligand. The effect of this hydrogen bond on the local structure of the [NiFe] active site is discussed.

Rhesus expression in a green alga is regulated by CO(2).

The function of the Rhesus (Rh) complex in the human red cell membrane has been unknown for six decades. Based on the organismal, organ, and tissue distribution of Rh proteins, and on our evidence that their only known paralogues, the ammonium and methylammonium transport proteins (also called methylammonium permeases), are gas channels for NH(3), we recently speculated that Rh proteins are biological gas channels for CO(2). Like NH(3), CO(2) differs from other gases in being readily hydrated. We have now tested our speculation by studying expression of the RH1 gene in the photosynthetic microbe Chlamydomonas reinhardtii. Expression of RH1 was high for cells grown in air supplemented with 3% CO(2) or shifted from air to high CO(2) (3%) for 3 h. Conversely, RH1 expression was low for cells grown in air (0.035% CO(2)) or shifted from high CO(2) to air for 3 h. These results make viable the hypothesis that Rh1 and Rh proteins generally are gas channels for CO(2).

Kinetic Mechanisms of IκB-related Kinases (IKK) Inducible IKK and TBK-1 Differ from IKK-1/IKK-2 Heterodimer

Nuclear factor-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB. The phosphorylation of IkappaBalpha on Ser(32) and Ser(36) is initiated by an IkappaB kinase (IKK) complex that includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK-1) and IkappaB kinase 2 (IKK-2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. Recently, two related IkappaB kinases, TBK-1 and IKK-i, have been described. TBK-1 and IKK-i show sequence and structural homology to IKK-1 and IKK-2. TBK-1 and IKK-i phosphorylate Ser(36) of IkappaBalpha. We describe the kinetic mechanisms in terms of substrate and product inhibition of the recombinant human (rh) proteins, rhTBK-1, rhIKK-I, and rhIKK-1/rhIKK-2 heterodimers. The results indicate that although each of these enzymes exhibits a random sequential kinetic mechanism, the effect of the binding of one substrate on the affinity of the other substrate is significantly different. ATP has no effect on the binding of an IkappaBalpha peptide for the rhIKK-1/rhIKK-2 heterodimer (alpha = 0.99), whereas the binding of ATP decreased the affinity of the IkappaBalpha peptide for both rhTBK-1 (alpha = 10.16) and rhIKK-i (alpha = 62.28). Furthermore, the dissociation constants of ATP for rhTBK-1 and rhIKK-i are between the expected values for kinases, whereas the dissociation constants of the IkappaBalpha peptide for each IKK isoforms is unique with rhTBK-1 being the highest (K(IkappaBalpha) = 69.87 microm), followed by rhIKK-i (K(IkappaBalpha) = 5.47 microm) and rhIKK-1/rhIKK-2 heterodimers (K(IkappaBalpha) = 0.12 microm). Thus this family of IkappaB kinases has very unique kinetic properties.

Identification of the Erythrocyte Rh Blood Group Glycoprotein as a Mammalian Ammonium Transporter

The Rh blood group proteins are well known as the erythrocyte targets of the potent antibody response that causes hemolytic disease of the newborn. These proteins have been described in molecular detail; however, little is known about their function. A transport function is suggested by their predicted structure and from phylogenetic analysis. To obtain evidence for a role in solute transport, we expressed Rh proteins in Xenopus oocytes and now demonstrate that the erythroid Rh-associated glycoprotein mediates uptake of ammonium across cell membranes. Rh-associated glycoprotein carrier-mediated uptake, characterized with the radioactive analog of ammonium [(14)C]methylamine (MA), had an apparent EC(50) of 1.6 mm and a maximum uptake rate (V(max)) of 190 pmol/oocyte/min. Uptake was independent of the membrane potential and the Na(+) gradient. MA transport was stimulated by raising extracellular pH or by lowering intracellular pH, suggesting that uptake was coupled to an outwardly directed H(+) gradient. MA uptake was insensitive to additions of amiloride, amine-containing compounds tetramethyl- and tetraethylammonium chloride, glutamine, and urea. However, MA uptake was significantly antagonized by ammonium chloride with inhibition kinetics (IC(50) = 1.14 mm) consistent with the hypothesis that the uptake of MA and ammonium involves a similar H(+)-coupled counter-transport mechanism.

Band 3 anion exchanger and its involvement in erythrocyte and kidney disorders.

Recent developments in the structure of erythrocyte band 3 and its role in hereditary spherocytosis and distal renal tubular acidosis are described. The crystal structure of the N-terminal cytoplasmic domain provides a basis for understanding the organization of ankyrin and other peripheral membrane proteins around band 3. Band 3 also binds integral membrane proteins, including the Rh protein complex and CD47. Band 4.2 is important in these associations, which link the Rh complex to the skeleton. It is suggested that band 3 forms the scaffold for a protein assembly that could transduce signals from the cell exterior and modulate the transport and mechanical properties of the erythrocyte. The involvement of band 3 in distal renal tubular acidosis is reviewed. The article discusses a likely mechanism for dominant distal renal tubular acidosis in which associations between the normal and mutant protein alter the plasma membrane targeting of the normal protein in the kidney.

Rh-deficiency syndrome

Jean-Pierre Cartron is Director of the Research Unit U76 of the Institut National de la Santé et de la Recherche Médicale, and Scientific Director of the Institut National de la Transfusion Sanguine in Paris.

Rhesus protein function emerges

The human rhesus (Rh) blood group system is of central importance in transfusion medicine and neonatal haematology. The Rh antigens involved in this system are expressed on the surface of erythrocytes and form a tetrameric complex of polytopic membrane proteins consisting of two Rh30 and two Rh50 proteins. Defects in these proteins lead to a number of genetic disorders that are grouped under the umbrella of Rh-deficiency syndrome. Since their discovery over 60 years ago, the nature of the Rh proteins has been determined at the genetic, immunological and biochemical level, but, apart from expressing antigens on their surface, the physiological roles of these proteins in the erythrocyte remain unclear. Anne-Marie Marini and Bruno Andre, from the Free University of Brussels, previously made the intriguing observation that the Rh proteins are homologous to the Mep/Amt family of ammonium transporters. In collaboration with the group of Jean-Pierre Cartron and Baya Chérif-Zahar from INSERM in Paris, they now demonstrate that the Rh50 protein from human erythrocytes (RhAG) can function as an ammonium transporter in a strain of the yeast Saccharomyces cerevisiae that lacks endogenous Mep/Amt proteins 1. Marini A-M et al. The human Rhesus-blood-group associated RhAG proteins and a novel kidney-specific homologue promote ammonium transport in yeast. Nat. Genet. 2000; 26: 341-344 Crossref PubMed Scopus (294) Google Scholar .

Localization of the Rh50-like protein to the contractile vacuole in Dictyostelium.

The human Rhesus (Rh) family consists of three polytopic membrane proteins present at the cell surface of red blood cells. Although Rh proteins are essential for the expression of the blood group system their biological function remains unclear. In this study, the gene encoding a protein homologous to Rh50 in Dictyostelium discoideum was sequenced. The Rh50-like protein was localized to the contractile vacuole, the organelle responsible for maintenance of osmotic equilibrium within the cell. However, Rh50-like-deficient mutants in which the Rh50-like gene was disrupted did not appear to exhibit a phenotype related to osmoregulation. Nevertheless, these mutants may provide a valuable tool for studying the function of the rhesus protein.

New Insights into the Rh Superfamily of Genes and Proteins in Erythroid Cells and Nonerythroid Tissues

ABSTRACTThe past decade has seen extensive studies of the erythrocyte Rh30 polypeptides and Rh-associated glycoprotein, which specify the clinically important Rh blood group system. Here we consider recent advances on these and other Rh homologues in the context of gene organization, molecular evolution, tissue-specific expression, protein structure, and potential biological functions. The Rh family is now known to contain a large number of homologues that form a unique branch in the eucarya life domain. The ancient origin and broad distribution imply central roles for the various Rh proteins in maintaining normal cellular and organismal homeostatic conditions. Rh homologues occur in the form of multiple chromosomal loci in mice and humans, but as single-copy genes in unicellular organisms (e.g., green alga and slime mold). While primitive Rh genes vary largely in exon/intron design, the mammalian Rh homologues bear a similar genomic organization. Sequence comparisons have revealed the signatures and a consensus 12-transmembrane fold characteristic of the Rh family. Phylogenetic analysis has placed all Rh homologues as a related cluster that intercepts ammonium transporter (Amt) clusters, indicating an intimate evolutionary and structural relationship between the Rh and Amt families. The biochemical identification and epithelial expression of RhBG and RhCG orthologues in mammalian kidney, liver, skin, testis, and brain suggest that they serve as transporters likely participating in ammonia homeostasis. Further inquires into the structure, function, biosynthesis, and interaction of Rh proteins will shed new light on ammonia homeostasis in a wide range of human physiological and pathological states.

Evolutionary history of the Rh blood group-related genes in vertebrates.

Rh and its homologous Rh50 gene products are considered to form heterotetramers on erythrocyte membranes. Rh protein has Rh blood group antigen sites, while Rh50 protein does not, and is more conserved than Rh protein. We previously determined both Rh and Rh50 gene cDNA coding regions from mouse and rat, and carried out phylogenetic analyses. In this study, we determined Rh50 gene cDNA coding regions from African clawed frog and Japanese medaka fish, and examined the long-term evolution of the Rh blood group and related genes. We constructed the phylogenetic tree from amino acid sequences. Rh50 genes of African clawed frog and Japanese medaka fish formed a cluster with mammalian Rh50 genes. The gene duplication time between Rh and Rh50 genes was estimated to be about 510 million years ago based on this tree. This period roughly corresponds to the Cambrian, before the divergence between jawless fish and jawed vertebrates. We also BLAST-searched an amino acid sequence database, and the Rh blood group and related genes were found to have homology with ammonium transporter genes of many organisms. Ammonium transporter genes can be classified into two major groups (amt alpha and amt beta). Both groups contain genes from three domains (bacteria, archaea, and eukaryota). The Rh blood group and related genes are separated from both amt alpha and beta groups.

Perinatal Consequences of Maternal-Fetal Rh Blood Group Interaction in Diabetic Pregnancy: A Nonimmunological Perspective

The recent discoveries about the structure of Rh protein that suggest a transport function and the recent observations of a positive correlation between Rh(D) protein and glycosylated hemoglobin levels in non-insulin-dependent diabetes mellitus prompted us to review our data on diabetic pregnancy to evaluate the perinatal consequences of maternal-fetal Rh blood group interactions in a metabolic perspective. One hundred thirty-two women with gestational diabetes and 120 women with preexisting insulin-dependent diabetes mellitus were examined. Three hundred eighty-seven consecutive nondiabetic puerperae from the same population were considered control subjects. In both gestational and insulin-dependent diabetes mellitus, an increased proportion of mother Rh(+)/newborn Rh(-) and a decreased proportion of mother Rh(-)/newborn Rh(+) joint phenotype has been observed. No deviation has been observed for joint phenotypes in which mother and newborn are similar [ie, Rh(+)/Rh(+) and Rh(-)/Rh(-)]. In the situation of mother Rh(+)/newborn Rh(-), there is a relatively lower rate of fetal loss and a decreased tendency to high birth weight. On the contrary, in pairs mother Rh(-)/newborn Rh(+) the fetus shows an increase of fetal loss and of tendency to high birth weight. The results are compatible with the hypothesis that when the density of Rh protein in the mother is higher than that in the fetus, the conceptus is relatively protected against the toxic effect of glucose. In the opposite genotypic combination (ie, density of Rh protein higher in the fetus than in the mother), the fetus is relatively more susceptible to these effects.

Perinatal Consequences of Maternal-Fetal Rh Blood Group Interaction in Diabetic Pregnancy: A Nonimmwnological Perspective

ABSTRACT Background The recent discoveries about the structure of Rh protein that suggest a transport function and the recent observations of a positive correlation between Rh(D) protein and glycosylated hemoglobin levels in non–insulin-dependent diabetes mellitus prompted us to review our data on diabetic pregnancy to evaluate the perinatal consequences of maternal-fetal Rh blood group interactions in a metabolic perspective. Subjects and Methods One hundred thirty-two women with gestational diabetes and 120 women with preexisting insulin-dependent diabetes mellitus were examined. Three hundred eighty-seven consecutive nondiabetic puerperae from the same population were considered control subjects. Results In both gestational and insulin-dependent diabetes mellitus, an increased proportion of mother Rh(+)/newborn Rh(−) and a decreased proportion of mother Rh(−)/newborn Rh(+) joint phenotype has been observed. No deviation has been observed for joint phenotypes in which mother and newborn are similar [ie, Rh(+)/Rh(+) and Rh(−)/Rh(−)]. In the situation of mother Rh(+)lnewborn Rh(−), there is a relatively lower rate of fetal loss and a decreased tendency to high birth weight. On the contrary, in pairs mother Rh(−)/newborn Rh(+) the fetus shows an increase of fetal loss and of tendency to high birth weight. Conclusions The results are compatible with the hypothesis that when the density of Rh protein in the mother is higher than that in the fetus, the conceptus is relatively protected against the toxic effect of glucose. In the opposite genotypic combination (ie, density of Rh protein higher in the fetus than in the mother), the fetus is relatively more susceptible to these effects.

Molecular biology and genetics of the Rh blood group system

The Rh (Rhesus) blood group system is the most complex of the known human blood group polymorphisms. The expression of its antigens is controlled by a two-component genetic system consisting of RH and RHAG loci, which encode Rh30 polypeptides and Rh50 glycoprotein, respectively. Over the past decade, there has been a rapid advance in knowledge of the biochemistry, molecular biology, and genetics of the Rh genes and proteins. The primary structures of D and CcEe antigens have become well understood and the molecular genetic basis of a vast array of phenotype polymorphisms has been delineated. The identification of various molecular defects associated with Rh deficiency syndrome clarifies the nature of the amorph, suppressor, and modifier genes. The observed mutation spectrum defines a basic set of components essential for Rh complex assembly in the erythrocyte membrane. The resulting molecular information, combined with new experimental tools, is helping to dissect the fine structure of Rh antigens in terms of epitope mapping. The discovery of novel Rh homologs in primitive organisms and in nonerythroid tissues opens new avenues of research beyond the scope of erythrocytes and Rh antigens. This review provides an update on the Rh family in antigen expression, phenotype diversity, and disease association.