The challenge of understanding ammonium homeostasis and the role of the Rh glycoproteins.

Rh glycoproteins belong to the superfamily of ammonium transporters, but until recent functional studies their functional role was unknown. This review focuses on the functional results obtained in our laboratory after the heterologous expression of RhAG (the erythroid Rh glycoprotein) and RhCG (an epithelial Rh glycoprotein). RhAG and RhCG were expressed in two different expression systems (HeLa cells and Xenopus laevis oocytes) that differed in their endogenous membrane permeabilities for NH3 and NH4+. To check if RhAG and RhCG are ammonium transporters, we measured intracellular pH changes in cells exposed to an ammonium-containing solution, and analyzed the ammonium-induced NH3 and NH4+ transmembrane fluxes in control versus transfected cells. We observed that RhAG and RhCG expression induced an enhancement of the ammonium-induced initial alkalinization (related to NH3 influx into the cell) and secondary acidification (related to NH4+ influx into the cell). Moreover, sub-millimolar ammonium concentrations induced inward currents in voltage-clamped RhAG- and in RhCG-expressing oocytes. Taken together, these results show not only that RhAG and RhCG are ammonium transporters, but also that they are promoting the transmembrane transport of NH3 and of NH4+. Data from our laboratory and from other groups raise several questions that are discussed.

The human Rhesus-associated RhAG protein and a kidney homologue promote ammonium transport in yeast.

The Rhesus blood-group antigens are defined by a complex association of membrane polypeptides that includes the non-glycosylated Rh proteins (RhD and RhCE) and the RHag glycoprotein, which is strictly required for cell surface expression of these antigens. RhAG and the Rh polypeptides are erythroid-specific transmembrane proteins belonging to the same family (36% identity). Despite their importance in transfusion medicine, the function of RhAG and Rh proteins remains unknown, except that their absence in Rh(null) individuals leads to morphological and functional abnormalities of erythrocytes, known as the Rh-deficiency syndrome. We recently found significant sequence similarity between the Rh family proteins, especially RhAG, and Mep/Amt ammonium transporters. We show here that RhAG and also RhGK, a new human homologue expressed in kidney cells only, function as ammonium transport proteins when expressed in yeast. Both specifically complement the growth defect of a yeast mutant deficient in ammonium uptake. Moreover, ammonium efflux assays and growth tests in the presence of toxic concentrations of the analogue methylammonium indicate that RhAG and RhGK also promote ammonium export. Our results provide the first experimental evidence for a direct role of RhAG and RhGK in ammonium transport. These findings are of high interest, because no specific ammonium transport system has been characterized so far in human.

The members of the RH gene family (RH50 and RH30) followed different evolutionary pathways.

The evolution of the RH gene family is characterized by two major duplication events, the first one originating the RH50 and RH30 genes and the second one giving rise to RHCE and RHD, the two paralogous RH30 genes which encode the Rh blood group antigens in human. The new sequence data obtained here for mouse RH50 and RH30 and for macaque RH50 allowed us to compare the evolutionary rates of the two genes and to show that RH50 evolved about 2.6 times more slowly than RH30 at nonsynonymous positions. This result implies that Rh50 proteins were evolutionarily more conserved compared to Rh30 polypeptides, thus being indicative of the functional significance of the former protein in species as distantly related as sponge and human. The duplication event leading to RH50 and RH30 genes was estimated to have occurred between 250 and 346 million years ago. Moreover, we could also estimate that the duplication event producing the RHCE and RHD genes occurred some 8.5 +/- 3.4 million years ago, in the common ancestor of human, chimpanzee, and gorilla. Interestingly, this event seems to coincide with the appearance in these species of a G-to-T mutation in the RH50 gene which created a stop codon in the corresponding transcript. This led to an Rh50 C-terminal cytoplasmic domain shorter than that found in orangutan and early primates.

Shift from Rh-positive to Rh-negative phenotype caused by a somatic mutation within the RHD gene in a patient with chronic myelocytic leukaemia.

We report a female patient whose Rh phenotype shifted from RhD-positive to RhD-negative over a 3-year period (1991-94), during which time she was treated with mastectomy (1992) and local irradiation for a low-grade recurrent breast cancer. She was diagnosed with chronic myeloid leukaemia in 1994, and has since then received chemotherapy. The patient was repeatedly typed as O, RhD-positive between 1965 and 1991 and was repeatedly found RhD-negative after 1994. Bcr-Abl transcripts typical of Ph1 chromosome were detected. Molecular analysis indicated that the patient was heterozygous at the RH locus, carrying one haplotype in which the RHD gene exhibited a single nucleotide deletion (G600) resulting in a frameshift and premature stop codon, and a normal RHCE gene (allele Ce). The second haplotype contained only the RHCE gene (allele ce) and was normal. Further analysis carried out on total leucocytes, purified neutrophils, EBV-lymphoblastoid cell line and cultured erythroblasts indicated that the G600 deletion was restricted to the myeloid lineage. No modification of other blood group antigens could be detected. These findings suggest a somatic mutation which most probably occurred in a stem cell common to the myeloid lineage.

Rh-deficiency of the regulator type caused by splicing mutations in the human RH50 gene.

The Rh polypeptides and the glycoproteins Rh50, CD47, LW, and glycophorin B, which interact in the red blood cell membrane to form a multisubunit complex, are lacking or are severely reduced in the Rh-deficiency syndrome. We previously reported that in several Rhnull patients the RH50 gene was altered at the coding sequence level, resulting in either a single amino acid substitution or the synthesis of a truncated polypeptide. In the present report, we have detected two mutations in the intronic region of the RH50 gene that identify a new molecular mechanism involved in Rh-deficiency. The first mutation affected the invariant G residue of the 3' acceptor splice-site of intron 6, causing the skipping of the downstream exon and the premature termination of translation. The second mutation occurred at the first base of the 5' donor splice-site of intron 1. Both these mutations were found in homozygote state. RNase protection assays demonstrated that the Rh50 mRNA level was strongly reduced or undetectable in the 3' and 5' splice mutants, respectively. The different mutations affecting the RH50 gene are indicative of an heterogeneous mutational pattern, which further supports the hypothesis that the lack of the Rh50 protein may prevent the assembly or transport of the Rh membrane complex to the red blood cell surface.

Insights into the structure and function of membrane polypeptides carrying blood group antigens.

In recent years, advances in biochemistry and molecular genetics have contributed to establishing the structure of the genes and proteins from most of the 23 blood group systems presently known. Current investigations are focusing on genetic polymorphism analysis, tissue-specific expression, biological properties and structure-function relationships. On the basis of this information, the blood group antigens were tentatively classified into five functional categories: (i) transporters and channels, (ii) receptors for exogenous ligands, viruses, bacteria and parasites, (iii) adhesion molecules, (iv) enzymes and, (v) structural proteins. This review will focus on selected blood groups systems (RH, JK, FY, LU, LW, KEL and XK) which are representative of these classes of molecules, in order to illustrate how these studies may bring new information on common and variant phenotypes and for understanding both the mechanisms of tissue specific expression and the potential function of these antigens, particularly those expressed in nonerythroid lineage.

Molecular defects of the RHCE gene in Rh-deficient individuals of the amorph type.

The deficiency of Rh proteins on the red blood cells from individuals of the Rhnull amorph type may be the result of homozygosity for a silent allele at the RH locus. This phenotype is also associated with the lack or reduced expression of glycoproteins (Rh50, CD47, LW, and glycophorin B), which interact with Rh polypeptides to form the multisubunit Rh membrane complex. In this study, we describe two molecular alterations affecting the RHCE gene in two unrelated Rhnull amorph individuals bearing Rh50 and CD47 normal transcripts. The first type of mutation, located at the donor splice-site in intron 4, induced the activation of two cryptic splice-sites within this intron and one such site in exon 4 that all generated aberrant transcripts. The second type of mutation affected the coding region and introduced a frameshift and a premature stop codon resulting in a shorter predicted protein (398 v 417 residues), including a completely different C-terminus of 76 amino acids. This suggests that protein folding and/or protein-protein interaction mediated by the C-terminal domain of the Rh proteins may play a role in the routing and/or stability of the Rh membrane complex.

A novel single missense mutation identified along the RH50 gene in a composite heterozygous Rhnull blood donor of the regulator type.

Rare individuals who lack all of the Rh blood group antigens are called Rhnull and may be classified as "regulator" or "amorph" types. The suppression of Rh antigen expression for regulator types may be attributed to mutations of the RH50 gene, which is independent of the RH locus. The RH50 gene encodes a glycoprotein that interacts with the Rh proteins to form a functional complex within the red blood cell membrane. This report describes an RH50 gene mutation for a previously unclassified Rhnull donor. Sequencing cDNA clones from Rh50 mRNA revealed a single base change (G836A) yielding a missense and nonconservative mutation (Gly279Glu) within a predicted hydrophobic domain for this membrane protein. Genomic DNA studies using polymerase chain reaction (PCR) restriction analysis and sequencing showed that the Rhnull propositus was a composite heterozygote for this mutation, carrying two alleles with the A and G at nucleotide 836, respectively. In contrast, cDNA studies showed that only the A836 sequence was present, suggesting that the second allele with G836 was apparently silent (no transcript detected). Family studies showed that the mutant RH50 allele (836A) was inherited maternally, whereas the silent RH50 allele (836G) was from paternal transmission. These findings provide further evidence that rare but diverse genetic alterations may occur along the RH50 gene where the Rhnull syndrome of the regulator type occurs. The single amino acid change (Gly to Glu) provides insight into the critical value of these residues for assembly of the Rh antigen complex within the membrane.

Organization of the human RH50A gene (RHAG) and evolution of base composition of the RH gene family.

Human Rh (rhesus) antigens are expressed in the red cell membrane as a multi-subunit complex, the central core of which is presumably composed of a tetramer made of two Rh and two Rh50 protein subunits. The interaction between Rh and Rh50 polypeptides is thought to be crucial to the correct assembly and transport of the complex to the cell surface. Here, we show that the human RH50A gene (RHAG) is composed of 10 exons whose size and exon/intron junctions are well conserved compared to those of the RH genes. We have also analyzed the RH50A 5' flanking region where the transcription initiation site has been identified. These results conclusively establish that the RH50A and RH genes do belong to the same gene family. Moreover, we show that the RH50A and RH genes are embedded in different compositional genomic contexts (i.e., different isochores) that are likely to drive the evolution of these genes, the base compositions (G + C content) of which differ drastically. Finally, we propose a scenario in which an RH50-like gene is likely to have played a founding role in the evolution of the RH gene family.

Phosphatidylserine exposure and aminophospholipid translocase activity in Rh-deficient erythrocytes.

Endogenous phosphatidylserine (PS) exposure and lipid transport activity have been investigated for seven unrelated cases of Rhnull erythrocytes. Endogenous PS exposure was measured by prothrombinase activity. Out of six cases studied, two Rhnull samples exhibited abnormal aminophospholipid exposure, as suggested by the measurement of a lower Km of factor Xa for prothrombin. Aminophospholipid translocase activity was measured through the transbilayer redistribution of spin-labelled analogues of phospholipids. Provided that incubation conditions allow the maintainance of intracellular ATP level, no difference was observed between Rhnull and control erythrocytes, clearly indicating that the aminophospholipid translocase and Rh polypeptides are different molecular species.

Characterization of the recombination hot spot involved in the genomic rearrangement leading to the hybrid D-CE-D gene in the D(VI) phenotype.

In the Caucasian population, the RH locus of RhD-positive individuals is composed of two homologous genes, RHD and RHCE, arranged in tandem but of a single gene, RHCE, in RhD-negative individuals. Many variants recently characterized carry rearranged RH genes, most often by an unidirectional segmental DNA-exchange (gene-conversion) event. In D(VI) variants of type II, RHD is a D-CE-D hybrid gene in which the DNA fragment carrying exons 4-6 has been replaced by the corresponding sequences from the RHCE gene. To identify precisely and characterize the two transition sites, we have studied, by both PCR and sequence analysis, a genomic region between the 3' end of intron 3 and exon 7 in normal RHCE and RHD genes as well as in D(VI) DNA. We show that the D-CE breakpoint is located in intron 3, within a 250-bp fragment comprising an Alu S sequence, and that the CE-D breakpoint lies within a 39-bp fragment in intron 6. This Alu S sequence (and the 100-bp region immediately downstream) most likely defines a recombination hot spot, since there lies also the 5' breakpoint of different rearrangement events leading to D-CE and CE-D transitions in hybrid D(VI),DFR and Dc-,R(N) gene complexes, respectively.

Candidate gene acting as a suppressor of the RH locus in most cases of Rh-deficiency.

The Rh antigen is a multi-subunit complex composed of Rh polypeptides and associated glycoproteins (Rh50, CD47, LW and glycophorin B); these interact in the red cell membrane and are lacking or severely reduced in Rhnull cells. As a result, individuals with Rhnull suffer chronic haemolytic anaemia known as the Rh-deficiency syndrome. Most frequently, Rhnull phenotypes are caused by homozygosity of an autosomal suppressor gene unlinked to the RH locus (Rhnull regulator or Rhmod types). We have analysed the genes and transcripts encoding Rh, CD47 and Rh50 proteins in five such unrelated Rhnull cases. In all patients, we identified alteration of Rh50--frameshift, nucleotide mutations, or failure of amplification--which correlated with Rhnull phenotype. We propose that mutant alleles of Rh50, which map to chromosome 6p11-21.1, are likely candidates for suppressors of the RH locus accounting for most cases of Rh-deficiency.

Molecular analysis of the structure and expression of the RH locus in individuals with D--, Dc-, and DCw- gene complexes.

Rh blood group antigens of the D, C/c, and E/e series are carried by at least three red cell membrane polypeptides encoded by two highly related genes, RHD and RHCE. Homozygous individuals carrying the D--, Dc-, and DCw- gene complexes are characterized by a total or partial lack of expression of the RHCE-encoded antigens. Analysis of the molecular genetic basis of these rare conditions indicates that complete or partial expression defect of Cc/Ee antigens result from different alterations at the RH locus, but not from gross deletions. No rearrangement or mutation of the RHCE gene could be detected in donors homozygous for the D-- complex, suggesting that the lack of the Cc and Ee antigens might result from a reduced transcriptional activity of the RHCE gene. The Dc- and DCw- gene complexes, however, exhibited an important rearrangement of the RHCE gene. Instead of the normal RHCE gene, both variants carried a hybrid RHCE-D-CE gene in which exons 4 to 9 (Dc- complex) and 2 (or 3) to 9 (DCw- complex) of the RHCE gene, respectively, have been substituted by the equivalent region of the RHD gene. These gene conversion events provide an explanation for the well-described abnormal antigen profiles associated with the Dc- and DCw- complexes, like the increased expression of RhD, the reduced expression of RhC/c or RhCw, and the absence of RhE/e.

Molecular characterization of the Rh-like locus and gene transcripts from the rhesus monkey (Macaca mulatta).

The human Rh blood group locus consists of two structurally related genes (D and CcEe) in Rh-positive haplotypes but a single gene (CcEe) in Rh-negative haplotypes. The genome of rhesus monkeys (Macaca mulatta), while not expressing any of the human Rh D, C, c, E, or e specificities, carries a Rh-like locus strongly related to the human Rh locus. Southern blot analysis suggested the presence of only one Rh-like gene with an additional truncated fragment corresponding to the 5' region. RNA preparations from M. mulatta bone marrow cells contained Rh-like species of 1.7 kb. Two allelic Rh-like transcripts were amplified by PCR and sequenced. The predicted translation product of the first transcript was a 417-amino-acid protein closely similar to the human Rh counterpart. The predicted product of the second transcript consisted of a 361-amino-acid polypeptide truncated in the NH2 terminal region and differing from the former by a few substitutions. The macaque Rh-like protein sequences differed from those of human D and Cc/Ee polypeptides by 22-25%, whereas the degree of identity between the human proteins was 91.5%. Implications of these results in the analysis of the evolutionary pathway of the Rh locus are discussed.

Organization of the gene (RHCE) encoding the human blood group RhCcEe antigens and characterization of the promoter region.

The human RH (rhesus) locus is composed of two genes, RHD and RHCE, encoding the D, Cc, and Ee blood group antigens. The RHCE gene was isolated from a human genomic library and characterized. It is organized into 10 exons distributed over 75 kb. Exons 4-8 are alternatively spliced in the different RNA isoforms previously identified. Primer extension analysis indicated that the transcription initiation site is located 83 bp upstream of the initiation codon. The 5' flanking region of the RHCE gene, from nucleotide -600 to +42, exhibited a significant transcriptional activity after transfection in the erythroleukemic cell line K562, but not in the nonhematopoietic cell line HeLa. This result was in agreement with Northern blot analysis, suggesting that the expression of the RH locus is restricted to the erythroid/megakaryocytic lineage. Accordingly, putative binding sites for SP1, GATA-1, and Ets proteins, nuclear factors known to be involved in the erythroid and megakaryocytic gene expression, were identified in this Rh promoter.

Chimpanzee Rh-like blood group genes map to chromosome region 1p36.1-->p34.2 by in situ hybridization.

A human Rh cDNA probe was used to map the Rh-like genes in the chimpanzee. The data gathered made it possible to uniquely localize these genes to chimpanzee chromosome region 1p36.1-->p34.2. This chromosomal localization is homologous to the location of the Rh genes in the human genome.

Molecular genetic basis of the human Rhesus blood group system.

The Rhesus (RH) blood group locus is composed of two related structural genes, D and CcEe, that encode red cell membrane proteins carrying the D, Cc and Ee antigens. As demonstrated previously, the RhD-positive/RhD-negative polymorphism is associated with the presence or the absence of the D gene. Sequence analysis of transcripts and genomic DNA from individuals that belong to different Rh phenotypes were performed to determine the molecular basis of the C/c and E/e polymorphisms. The E and e alleles differ by a single nucleotide resulting in a Pro226Ala substitution, whereas the C and c alleles differ by six nucleotides producing four amino acid substitutions Cys16Trp, Ile60Leu, Ser68Asn and Ser103Pro. With the recent cloning of the RhD gene, these findings provide the molecular genetic basis that determine D, C, c, E and e specificities.