Disruption of a GATA motif in the Duffy gene promoter abolishes erythroid gene expression in Duffy-negative individuals.

The mRNA for the Duffy blood group antigen, the erythrocyte receptor for the Plasmodium vivax malaria parasite, has recently been cloned and shown to encode a widely expressed chemokine receptor. Here, we show that the Duffy antigen/chemokine receptor gene (DARC) is composed of a single exon and that most Duffy-negative blacks carry a silent FY*B allele with a single T to C substitution at nucleotide -46. This mutation impairs the promoter activity in erythroid cells by disrupting a binding site for the GATA1 erythroid transcription factor. With the recent characterization of the FY*A and FY*B alleles, these findings provide the molecular basis of the Duffy blood group system and an explanation for the erythroid-specific repression of the DARC gene in Duffy-negative individuals.

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.

Role of Lu/BCAM in abnormal adhesion of sickle red blood cells to vascular endothelium.

Lutheran (Lu) blood group and Basal Cell Adhesion Molecule (BCAM) antigens are both carried by two glycoprotein (gp) isoforms of the immunoglobulin superfamily representing receptors for laminin alpha5 chain. They are expressed in red blood cells, in endothelial cells of vascular capillaries and in epithelial cells of several tissues. Lu/BCAM gps are overexpressed in sickle red blood cells (SS RBCs). Stimulation of SS RBCs by epinephrine activates the PKA depending signaling pathway and induces reinforced Lu/BCAM-mediated adhesion to laminin10/11. We have analyzed the phosphorylation state of Lu/BCAM long isoform cytoplasmic tail and showed that it is phosphorylated by CKII, GSK3b and PKA. Phosphorylation of this isoform in transfected K562 cells is stimulated by effectors of the PKA pathway and induces cell adhesion to laminin10/11. Lu/BCAM gps are highly expressed in endothelial cells and exhibit potential integrin binding motifs. We showed that they interact with integrin alpha4beta1, the unique integrin expressed on the surface of young reticulocytes. Adhesion assays under flow conditions showed that SS RBCs adhere to primary human endothelial cells (HUVEC) after selective activation of intergin alpha4beta1 and that this adhesion is mediated by endothelial Lu/BCAM gps. Our studies show that Lu/BCAM gps expressed either on erythroid or on endothelial cells are involved in SS RBC-endothelium interactions and could play a role in the abnormal adhesion of SS RBCs to vascular endothelium contributing to the vaso-occlusive crises reported for sickle cell disease patients.

Red cell and endothelial Lu/BCAM beyond sickle cell disease.

Recent studies shed new lights on the biological function of blood group antigens, such as the adhesion properties of the Lutheran (Lu) blood group antigens carried by the Lu/BCAM glycoproteins. The Lu/BCAM adhesion glycoproteins were first identified as laminin-10/11 erythroid receptors involved in RBC adhesion to endothelium in sickle cell anemia. Lu/BCAM mediated cell adhesion to laminin is stimulated by epinephrine, a physiological stress mediator, and is dependent of phosphorylation by protein kinase A. More recently, we demonstrated that constitutive phosphorylation of Lu/BCAM is also involved in abnormal RBC adhesion to endothelium in patients with polycythemia vera (PV), a frequent myeloproliferative disorders associated with the V617F mutation of the tyrosine kinase JAK2 leading to continuous stimulation of erythropoiesis. This observation suggests that Lu/BCAM could participate to the high incidence of vascular thrombosis that also characterizes PV disease. In mice, which do not express Lu/BCAM in erytroid tissues, invalidation of the Lu/BCAM gene provided evidence that Lu/BCAM gps, as laminin-alpha5 receptors, are involved in vivo in the maintenance of normal basement membrane organization in different non erythroid tissues since up to 90% of the mutant kidney glomeruli exhibited a reduced number of visible capillary lumens and irregular thickening of the glomerular basement membrane, while intestine exhibited smooth muscle coat thickening and disorganization. All these results further illustrate that minor blood group antigens might have important role under physiological and physiopathological conditions in erythroid and non erythroid tissues as well.

Noninvasive fetal RHD genotyping from maternal plasma. Use of a new developed Free DNA Fetal Kit RhD.

Fetal RHD genotyping from maternal plasma was performed by real-time PCR amplification of exons 7 and 10 of the RHD gene and the amplified products were detected either with SYBR Green I dye according to our previously published method [Mol Diagn 8 (2004) 23-31] or with hydrolysis probes in a new Free DNA Fetal Kit RhD((R)). Plasma specimen from 300 RhD-negative pregnant women (between 10 to 34 weeks of gestation) were analysed and validation of the results was ascertained either by RHD genotyping on amniotic cells or by blood typing of the neonate at birth. We found 100% concordant results when comparing the two methods. Two false-positive but no false-negative results were found. Thus, the sensitivity of the assay was 100% and the specificity superior than 99%. These data confirm the accuracy of fetal RHD genotyping on maternal plasma using the Free DNA Fetal Kit RhD, thus allowing to propose non invasive PCR-based fetal RHD genotyping for all RhD-negative pregnant women and to restrict the use of anti-D immunoglobulins only to those bearing an RhD-positive fetus.

A structural model of a seven-transmembrane helix receptor: the Duffy antigen/receptor for chemokine (DARC).

The Duffy antigen/receptor for chemokine (DARC) is an erythrocyte receptor for malaria parasites (Plasmodium vivax and Plasmodium knowlesi) and for chemokines. In contrast to other chemokine receptors, DARC is a promiscuous receptor that binds chemokines of both CC and CXC classes. The four extracellular domains (ECDs) of DARC are essential for its interaction with chemokines, whilst the first (ECD1) is sufficient for the interaction with malaria erythrocyte-binding protein. In this study, we elaborate and analyze structural models of the DARC. The construction of the 3D models is based on a comparative modeling process and on the use of many procedures to predict transmembrane segments and to detect far homologous proteins with known structures. Threading, ab initio, secondary structure and Protein Blocks approaches are used to build a very large number of models. The conformational exploration of the ECDs is performed with simulated annealing. The second and fourth ECDs are strongly constrained. On the contrary, the ECD1 is highly flexible, but seems composed of three consecutive regions: a small beta-sheet, a linker region and a structured loop. The chosen structural models encompass most of the biochemical features and reflect the known experimental data. They may be used to analyze functional interaction properties.

Ammonium transport properties of HEK293 cells expressing RhCG mutants: preliminary analysis of structure/function by site-directed mutagenesis.

We have recently shown by monitoring intracellular pHi with a stopped-flow fluorimeter, that when expressed in HEK293 kidney cells, two Rh glycoproteins, RhBG and RhCG, facilitated NH3 movement across the plasma membrane. Based on the results of 3D structure determination of AmtB, a bacterial member of the Amt/Mep/Rh superfamily, and of homology modeling of the human Rh proteins, we have attempted to determine if some selected residues predicted to be located in the pore or in the vestibule of the channel are essential for NH3 transport. Accordingly, wild type and mutant forms of RhCG were expressed in HEK293 cells and their ammonium function was analyzed with the stopped-flow fluorimeter. Some mutants that were not expressed at a significant level in HEK293 could not be tested for function, but mutations at positions F74, V137 and F235 (equivalent positions in AmtB: I28, L114, F215, respectively) resulted in a severe reduction of NH3 transport.

Functional interaction between Rh proteins and the spectrin-based skeleton in erythroid and epithelial cells.

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.

Early expression of glycophorin C during normal and leukemic human erythroid differentiation.

Glycophorins C and D (GPC and GPD) are two erythrocyte glycoproteins which originate from the same gene but differ in their NH2-terminal residues. The cell surface expression of these glycoproteins during normal and erythroid differentiation has been investigated with monoclonal and polyclonal antibodies and has been compared to the expression of glycophorin A (GPA), the major sialoglycoprotein of human red cells. Using glycosylation-independent antibodies (monoclonal or polyclonal), GPC or GPD was detected in erythroid and nonerythroid cell lineages. However, a glycosylation-dependent monoclonal antibody (MR4-130) detected an epitope on GPC which appears to be erythroid specific, suggesting that lineage specificity of this glycoprotein is related to some carbohydrate structures. During normal erythroid differentiation, GPC was expressed early at the level of erythroid progenitors (part of erythroid burst-forming unit and erythroid colony-forming unit) as detected with a glycosylation-independent monoclonal antibody (APO 3), whereas GPA is only present during terminal erythroid differentiation. The MR4-130 epitope was not coordinately expressed on the cell surface with the GPC molecule in the erythroid differentiation, since it was detected at the level of the more mature erythroid colony-forming unit slightly later than the GPC polypeptide. In four erythroleukemic patients, blast cells blocked at discrete stages of the erythroid differentiation were also investigated with antibodies and complementary DNA probes for GPA and GPC. GPA was immunologically detected in three of four cases, and its cell surface expression was correlated with the amount of specific mRNA in the cells, as seen by Northern blot analysis. GPC was immunologically detected on the blast cells of all four patients. However, in two cases including one with positive expression of GPA, the MR4-130 epitope was absent from the GPC molecule. By Northern blot analysis, we found that the GPC/GPD mRNA was present at a high level in all four patient samples. Western blot analysis of GPC and GPD in two of these patients revealed that these mRNAs were mostly translated into the GPD molecule, suggesting that these glycoproteins might be differently processed in certain cases of erythroleukemia.

TSPO ligands stimulate ZnPPIX transport and ROS accumulation leading to the inhibition of P. falciparum growth in human blood.

After invading red blood cells (RBCs), Plasmodium falciparum (Pf) can export its own proteins to the host membrane and activate endogenous channels that are present in the membrane of RBCs. This transport pathway involves the Voltage Dependent Anion Channel (VDAC). Moreover, ligands of the VDAC partner TranSlocator PrOtein (TSPO) were demonstrated to inhibit the growth of the parasite. We studied the expression of TSPO and VDAC isoforms in late erythroid precursors, examined the presence of these proteins in membranes of non-infected and infected human RBCs, and evaluated the efficiency of TSPO ligands in inhibiting plasmodium growth, transporting the haem analogue Zn-protoporphyrin-IX (ZnPPIX) and enhancing the accumulation of reactive oxygen species (ROS). TSPO and VDAC isoforms are differentially expressed on erythroid cells in late differentiation states. TSPO2 and VDAC are present in the membranes of mature RBCs in a unique protein complex that changes the affinity of TSPO ligands after Pf infection. TSPO ligands dose-dependently inhibited parasite growth, and this inhibition was correlated to ZnPPIX uptake and ROS accumulation in the infected RBCs. Our results demonstrate that TSPO ligands can induce Pf death by increasing the uptake of porphyrins through a TSPO2-VDAC complex, which leads to an accumulation of ROS.

A comparative study of the ori sequences from the mitochondrial genomes of twenty wild-type yeast strains.

The ori sequences of the mitochondrial genomes of 20 wild-type strains of Saccharomyces cerevisiae were compared with those of the previously studied strain A (de Zamaroczy et al., 1984). The seven canonical ori sequences of this strain appear to be present in all strains tested, but in most strains ori1 is replaced by an extensively rearranged ori1 * sequence, and an additional ori sequence, ori8, is present between the oxi3 and the 15S RNA genes; one strain, B, lacks ori4. The location and orientation of ori sequences of three strains, B, C and K, were found to be the same as in strain A. The primary structures of four ori sequences from three different strains (ori1 of strain J69-1B, ori3 and ori5 of strain K, ori6 of strain D273-10B) were found to be identical with the corresponding ori sequences previously investigated. Hybridization experiments with different ori probes indicated a conservation of ori2-ori7 sequences in all strains tested. The primary structure of a petite genome derived from strain B and carrying ori1 * is reported and discussed.

Tentative model for the mapping of D epitopes on the RhD polypeptide.

The partial D phenotypes correspond to D-positive individuals that may develop anti-D antibodies following immunization by transfusion or pregnancy, since they lack some of the D epitopes that compose the D antigen. When these red cells are tested with a panel of human monoclonal anti-D, different patterns of reactivity are observed and at least nine distinct epitopes termed epD1 to epD9 can be identified. Molecular analysis of partial D variants have shown that the loss of some D epitopes is associated either with intergenic recombination events between the D and CE genes generating hybrid gene structures D-CE-D or CE-D-CE, or with point mutations of the D gene. Based on these findings, a tentative model that correlates critical amino acid positions and D epitope expression on the D protein was proposed. Although recent studies suggest that the D antigen may be composed of as many as 30 epitopes, the relatively simple model presented here may be useful to serologists as a preliminary approach to understanding the basis of D antigenic variation in terms of structure-activity relationship.

Role of Lu/BCAM glycoproteins in red cell diseases.

Lu/BCAM glycoproteins (gps) are the unique erythroid receptors of laminin alpha5 chain, a major component of the extracellular matrix. They interact with the membrane skeleton by binding directly to spectrin via the Lu/BCAM RK573-574 motif. Lu/BCAM gps are involved in abnormal sickle red blood cell (RBC) adhesion to components of the vascular wall. This adhesion is activated by the phosphorylation of the Lu/BCAM long isoform Lu in a protein kinase A-dependent manner. A similar high adhesion to laminin was also observed with RBCs from Hereditary Spherocytosis (HS) patients suffering from haemolytic anaemia subsequent to spectrin deficiencies. We investigated the molecular mechanisms responsible for the Lu/BCAM-mediated abnormal RBC adhesion to laminin in sickle cell disease (SCD) and HS. We showed that SCD patients treated with hydroxycarbamide (HC) had a diminished RBC adhesion to laminin that was associated with reduced levels of the PKA upstream effector cAMP and a severe decrease in Lu isoform phosphorylation. On the other hand, we showed that increased Lu/BCAM-mediated HS RBC adhesion to laminin was independent of Lu/BCAM phosphorylation. A cellular model expressing the RK573-574AA Lu/BCAM mutant, which is unable to bind to spectrin, showed increased Lu/BCAM detergent extractability and enhanced cell adhesion to laminin. Similar results were obtained with HS RBCs, strongly suggesting that their increased adhesion could result from alteration of the Lu/BCAM-spectrin interaction following the severe spectrin deficiency.

Recent advances in molecular and genetic analysis of Rh blood group structures.

The Rh antigens are carried by 30-32 kDa integral proteins of the red cell membrane. The Rh locus is composed of two genes: RhD, which encodes the major D antigen and is present only in Rh-positive genomes, and RhCcEe, which encodes both the Cc and Ee polypeptides, most likely by alternative splicing events. The D and non-D Rh mRNAs have been cloned. Their sequence homology suggest that the two Rh genes have evolved by duplication of a common ancestor.

Molecular basis for elliptocytosis associated with glycophorin C and D deficiency in the Leach phenotype.

Glycophorin C (GPC) and glycophorin D (GPD) are highly glycosylated integral membrane proteins of human erythrocytes encoded by the same gene and associated with expression of Gerbich blood group system antigens. GPC/D deficiency (the Leach phenotype) is a rare condition usually found after identification of Gerbich blood group system antibodies in persons undergoing prenatal or pretransfusion evaluation. In all cases, the Leach phenotype has been associated with elliptocytosis. Characterization of the molecular basis of this phenotype in three previously uninvestigated families has shown that the most common genetic basis of GPC/D deficiency is deletion of exons 3 and 4 of the GPC gene. However, in one family, the Leach phenotype appeared due to a deletion of one nucleotide in exon 3, causing a frameshift mutation in the messenger RNA and premature generation of a stop codon. The GPC and GPD protein sequences are therefore interrupted in the extracellular domain and probably intracellularly degraded.

Molecular basis and PCR-DNA typing of the Fya/fyb blood group polymorphism.

The Duffy blood group antigens are carried by the erythrocyte membrane glycoprotein gpD, which has a molecular weight of 35-45 kDa and which has been recently cloned. In this report, we have determined, at the nucleic acid level, the molecular basis for the blood group Fya/Fyb polymorphism. The gpD cDNAs isolated by reverse transcription/polymerase chain reaction (RT-PCR) from Fy(a+b-) and Fy(a-b+) donors differed by only one base substitution (G131A) changing Gly to Asp at position 44 of the gpD protein. When expressed in simian Cos-7 cells, the Gy(a+b-) and Fy(a-b+) gpD cDNA produce cell surface proteins that react with the anti-Fya and anti-Fyb antisera, respectively, demonstrating that they represent the FY*A and FY*B alleles of the Duffy blood group locus. The G131A nucleotide substitution has been correlated with a BanI restriction site polymorphism, which has allowed us to develop a method for the DNA typing of the main Duffy blood group antigens, by means of PCR/restriction fragment length polymorphisms.

Leu110Pro substitution in the RhD polypeptide is responsible for the DVII category blood group phenotype.

The nucleotide sequence of the RhD transcripts from the reticulocytes of three unrelated variants with the DVII category blood group phenotype has been determined. Our results indicate that the expression of the low frequency antigen Rh40 and the lack of epD8 at the surface of these variant RhD positive red cells are associated with a single point mutation, T329C, in exon 2 of the RHD gene. This nucleotide polymorphism results in a leucine to proline substitution at amino acid position 110 of the RhD polypeptide.

A unique gene encodes spliceoforms of the B-cell adhesion molecule cell surface glycoprotein of epithelial cancer and of the Lutheran blood group glycoprotein.

Two new members of the Ig superfamily, the Lutheran (Lu) blood group glycoprotein and the B-cell adhesion molecule (B-CAM) epithelial cancer antigen, have been recently cloned from human placenta and colon cancer HT29 cell line, respectively. Although amino acid sequences deduced from cDNA analysis suggested that B-CAM should represent an abridged form of the Lu glycoprotein lacking the last 40 amino acids of the putative cytoplasmic tail, the relationship between the genes encoding these polypeptides has not been determined. In the present report, we showed by Southern blot analysis that the Lu and B-CAM cDNAs derived from a unique LU gene which exhibited an HindIII RFLP associated with the Lua/Lub blood group polymorphism. Accordingly, in situ hybridization of the Lu cDNA probe confirmed the localization of the Lutheran blood group locus to chromosome 19 q13.2-13.3, as previously shown for a B-CAM DNA probe. Sequence comparison between cDNA and genomic PCR fragments indicated that the Lu and B-CAM transcripts previously isolated are generated through the alternative use of internal splice donor and acceptor sites within an exon located at the 3' end of the LU gene. These spliceoforms corresponded to 2.5 kb and 4.0 kb mRNA species detectable by Northern blot in all tissues and cell lines in which the LU gene is expressed; their primary structures are consistent with the presence of both the Lu and B-CAM antigens on two glycoprotein isoforms. However, the 4.0 kb transcript was very poorly expressed as compared to the 2.5 kb species except in the colon carcinoma HT29 cell line, suggesting a differential regulation of the Lu/B-CAM messenger RNA in some tumor tissues.