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.

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.

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.

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.

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.

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.

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.

Structural characterization of the epitope recognized by the new anti-Fy6 monoclonal antibody NaM 185-2C3.

The epitope recognized by a new anti-Fy6 monoclonal antibody (MoAb) (clone name: NaM185-2C3) was characterized using peptides synthesized on pins (Epitope scanning kit). The clone was obtained from splenocytes of mice immunized with CHO cells expressing the recombinant Duffy glycoprotein. NaM185-2C3 recognized a linear epitope, the essential portion of which was pentapeptide Phe-Glu-Asp-Val-Trp comprising amino acid residues 22-26 of the main (336aa) isoform of the Duffy antigen. All the amino acid residues of the epitope, except Asp, were essential for the antibody-binding, because they could not be replaced by any or most other amino acid residues. The Asp residue could be replaced by most other amino acid residues and its replacement by some amino acid residues gave a distinct increase in the antibody-binding. The MoAb NaM185-2C3, similarly as other anti-Fy6 antibodies, inhibits interleukin (IL)-8-binding to the Duffy antigen. A part of the results was presented at ISBT meeting (Blanchard et al., 1998, Vox Sanguinis, 74, S1, Abstract no. 71).

Flow cytometric analysis of the association between blood group-related proteins and the detergent-insoluble material of K562 cells and erythroid precursors.

The linkage between blood group-related cell surface proteins and the detergent-insoluble material (DIM) was estimated by flow cytometry using a panel of specific monoclonal antibodies (mAbs) as a comparison of the antibody-binding capacity of intact and Triton-X100-treated cells. Studies were performed with K562 cells expressing endogenous or recombinant proteins and with human erythroid progenitors during their proliferation and differentiation in vitro. Glycophorin C (GPC) was found to be Triton-insoluble in both cellular models. When expressed (erythroid progenitors), Band 3 remained Triton-insoluble. Glycophorin A (GPA), however, behaved as Triton-soluble or insoluble according to the absence (K562) or the presence (erythroid progenitors) of Band 3 respectively. Comparison of the cellular models regarding the proteins that compose the Rh complex also indicated that Rh(D), RhAG and CD47 were resistant to Triton extraction in cells lacking Band 3. Similarly, RhAG and CD47 remained predominantly Triton-insoluble in K562 cells and early progenitors before Rh and Band 3 expression. Further analysis showed that the Kell protein was DIM-associated. In contrast, CD99 and DARC (Fy) proteins were not, or were very poorly, DIM-associated. Additionally, the adhesion molecules CD44 and Lu were completely or partially resistant to detergent extraction respectively. Deletion of the Lu cytoplasmic tail or its replacement by the cytoplasmic domain of GPC resulted in significant increase or decrease of the Triton solubility of the transfected proteins respectively. These data suggest that Triton insolubility of Lu results in part from direct attachment of its cytoplasmic tail with the cytoskeleton. We assume that this method should provide a useful tool to map interaction sites localized in the cytoplasmic domain of recombinant transmembrane proteins.

Isoforms of the Lutheran/basal cell adhesion molecule glycoprotein are differentially delivered in polarized epithelial cells. Mapping of the basolateral sorting signal to a cytoplasmic di-leucine motif.

Lu and Lu(v13) are two glycoprotein (gp) isoforms that belong to the immunoglobulin superfamily and carry both the Lutheran (Lu) blood group antigens and the basal cell adhesion molecule epithelial cancer antigen. Lu (85 kDa) and Lu(v13) (78 kDa) gps, which differ only in the length of their cytoplasmic domain, are adhesion molecules that bind laminin. In nonerythroid tissues, the Lu/basal cell adhesion molecule antigens are predominantly expressed in the endothelium of blood vessel walls and in the basement membrane region of normal epithelial cells, whereas they exhibit a nonpolarized expression in some epithelial cancers. Here, we analyzed the polarization of Lu and Lu(v13) gps in epithelial cells by confocal microscopy and domain-selective biotinylation assays. Differentiated human colon carcinoma Caco-2 cells exhibited a polarized expression of endogenous Lu antigens associated with a predominant expression of the Lu isoform at the basolateral domain of the plasma membrane and a very low expression of the Lu(v13) isoform at both the apical and basolateral domains. Analysis of transfected Madin-Darby canine kidney cells revealed a basolateral expression of Lu gp and a nonpolarized expression of Lu(v13) gp. Delivery of Lu(v13) to both apical and basolateral surfaces showed similar kinetics, indicating that this isoform is directly transported to each surface domain. A dileucine motif at position 608-609, specific to the Lu isoform, was characterized as a dominant basolateral sorting signal that prevents Lu gp from taking the apical delivery pathway.

Characterization of a mouse liminin receptor gene homologous to the human blood group Lutheran gene.

The human Lutheran (Lu) blood group antigens are carried by two glycoproteins (gps) that belong to the immunoglobulin (Ig) superfamily. These gps represent adhesion molecules that function as the unique erythroid receptors for laminin. We report here the cloning and functional expression of the orthologous mouse Lu mRNA as well as the genomic organization of the mouse Lu gene. The deduced human and mouse Lu gps share 72.5% identity and similar organization of the Ig-like domains. As in the human, the mouse Lu gene is organized in 15 exons. The proximal promoter showed consensus CACC-binding sites whereas the distal promoter exhibits a GATA-1-binding site and multiple E boxes. Like the human gene, the mouse Lu gene is also widely expressed among tissues but is transcribed as a unique 2.4-kb mRNA species. Expression of the mouse Lu mRNA is upregulated upon dimethyl sulfoxide-induced erythroid differentiation of murine erythroleukemia cells (MEL). During mouse embryonic development, the Lu transcript is detected as early as day 7 of gestation. Analysis of transfected human erythroleukemia K562 cells indicated that the adhesive properties of the Lu gps to laminin are conserved between human and mouse.

Arg89Cys substitution results in very low membrane expression of the Duffy antigen/receptor for chemokines in Fy(x) individuals.

The Duffy (FY) blood group antigens are carried by the DARC glycoprotein, a widely expressed chemokine receptor. The molecular basis of the Fya/Fyb and Fy(a-b-) polymorphisms has been clarified, but little is known about the Fyx antigen and the FY*X allele associated with weak expression of Fyb, Fy3, Fy5, and Fy6 antigens. We analyzed here the structure and expression of the FY gene in 4 Fy(a-bweak) individuals. As compared with Fy(a-b+) controls, the Fy(a-bweak) red blood cell membranes contained residual amount of DARC polypeptide and these cells were poorly bound by anti-Fy antibodies and chemokines. The FY gene from Fy(a-b+) and Fy(a-bweak) individuals differed by one substitution, C286T. The resulting Arg89Cys amino acid change reduced the binding of anti-Fy antibodies and chemokines to DARC transfectants. We concluded that the Fybweak donors carried the FY*X allele at the FY locus and that the Fyx antigen corresponds to highly reduced expression of a grossly normal Fyb polypeptide caused by the Arg89Cys substitution. Because FY is a single copy gene, this defect should also affect DARC expression in nonerythroid cells. Because the Fyx phenotype is not associated with apparent clinical consequences, we discussed these findings in the light of the putative roles of DARC in various tissues. Finally, we developed a Fyx DNA typing assay that should be useful for genetic studies and clinical transfusion medicine.

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.

The Lutheran blood group glycoproteins, the erythroid receptors for laminin, are adhesion molecules.

The Lutheran antigens are recently characterized glycoproteins in which the extracellular region contains five immunoglobulin like domains, suggesting some recognition function. A recent abstract suggests that the Lutheran glycoproteins (Lu gps) act as erythrocyte receptors for soluble laminin (Udani, M., Jefferson, S., Daymont, C., Zen, Q., and Telen, M. J. (1996) Blood 88, Suppl. 1, 6 (abstr.)). In the present report, we provided the definitive proof of the laminin receptor function of the Lu gps by demonstrating that stably transfected cells (murine L929 and human K562 cell lines) expressing the Lu gps bound laminin in solution and acquired adhesive properties to laminin-coated plastic dishes but not to fibronectin, vitronectin, transferrin, fibrinogen, or fibrin. Furthermore, expression of either the long-tail (85 kDa) or the short-tail (78 kDa) Lu gps, which differ by the presence or the absence of the last 40 amino acids of the cytoplasmic domain, respectively, conferred to transfected cells the same laminin binding capacity. We also confirmed by flow cytometry analysis that the level of laminin binding to red cells is correlated with the level of Lu antigen expression. Indeed, Lunull cells did not bind to laminin, whereas sickle cells from most patients homozygous for hemoglobin S overexpressed Lu antigens and exhibited an increased binding to laminin, as compared with normal red cells. Laminin binding to normal and sickle red cells as well as to Lu transfected cells was totally inhibited by a soluble Lu-Fc chimeric fragment containing the extracellular domain of the Lu gps. During in vitro erythropoiesis performed by two-phase liquid cultures of human peripheral blood, the appearance of Lu antigens in late erythroid differentiation was concomitant with the laminin binding capacity of the cultured erythroblasts. Altogether, our results demonstrated that long-tail and short-tail Lu gps are adhesion molecules that bind equally well laminin and strongly suggested that these glycoproteins are the unique receptors for laminin in normal and sickle mature red cells as well as in erythroid progenitors.

Specificity and sensitivity of RHD genotyping methods by PCR-based DNA amplification.

We have compared the sensitivity and specificity of four PCR methods of RHD gene detection using different sets of primers located in the regions of highest divergence between the RHD and RHCE genes, notably exon 10 (method I), exon 7 (method II), exon 4 (method III) and intron 4 (method IV). Methods I-III were the most sensitive and gave a detectable signal with D-pos/D-neg mixtures containing only 0.001% D-positive cells. Moreover, method II could detect the equivalent DNA amount present in only three nucleated cells in the assay without hybridization of PCR products, whereas the sensitivity of the other methods was 10-50 times less. Investigation of D variants indicated that false-negative results were obtained with method II (D(IVb) variant), method III (D(VI) and DFR variants) and method IV (D(VI) variants), but not method I. Weak D (D(u)) was correctly detected as D-positive by all methods, but most cases of Rh(null) appeared as false-positives, as they carry normal RH genes that are not phenotypically expressed. Some false-positive results were obtained with method I in a few Caucasian DNA samples serotyped as RhD-neg but carrying a C- or E-allele, whereas a high incidence of false-positives was found among non-Caucasian Rh-negative samples by all methods. In the Caucasian population, however, we found a full correlation between the predicted genotype and observed phenotype at birth of 92 infants. Although we routinely use the four methods for RHD genotyping, a PCR strategy based on at least two methods is recommended.

Organization of the human LU gene and molecular basis of the Lu(a)/Lu(b) blood group polymorphism.

The Lutheran (Lu) blood group antigens and the B-cell adhesion molecule (B-CAM) epithelial cancer antigen are carried by recently cloned integral glycoproteins that belong to the Ig superfamily. We have previously shown that the Lu and B-CAM antigens are encoded by the same gene, LU, and that alternative splicing of the primary transcript most likely accounts for the presence of both antigens on two isoforms that differ by the length of their cytoplasmic tails. In the present report, we isolated the human LU gene by cloning a 20-kb HindIII fragment from Lu(a - b+) genomic DNA. The LU gene is organized into 15 exons distributed over 12.5 kb. Alternative splicing of intron 13 generates the 2.5- and 4.0-kb transcript spliceoforms encoding the long tail and the short tail Lu polypeptides, respectively. Sequencing of the major mRNA species (2.5 kb) amplified from human bone marrow, kidney, placenta, and skeletal muscle did not suggest the presence of tissue-specific Lu glycoprotein isoforms. The same transcription initiation point, located 22 bp upstream from the initiation codon, was characterized in several tissues. In agreement with the wide tissue distribution of the Lu messengers, the GC-rich proximal 5' flanking region of the LU gene does not contain TATA or CAAT boxes, but includes several potential binding sites for the ubiquitous Sp1 transcription factor. In addition, the distal 5' region, encompassing nucleotides -673 to -764, contains clustered binding sequences for the GATA, CACCC, and Ets transcription factors. Analysis of the coding sequences amplified from genomic DNA of Lu(a + b-) or Lu(a - b+) donors showed a single nucleotide change in exon 3 (A229G) that correlates with an Aci I restriction site polymorphism and results in a His77Arg amino-acid substitution. Polymerase chain reaction/restriction fragment length polymorphism analysis indicated that the A229G mutation is associated with the Lu(a)/Lu(b) blood group polymorphism. When expressed in Chinese hamster ovary (CHO) cells, Lu cDNAs carrying the A229 or the G229 produced cell surface proteins that reacted with anti-Lu(a) or anti-Lu(b) antibodies, respectively, showing that these nucleotides specify the Lu(a) and Lu(b) alleles of the Lutheran blood group locus. CHO cells expressing recombinant short-tail or long-tail Lu glycoproteins reacted as well with anti-Lu as with anti-B-CAM antibodies, providing the definitive proof that the Lu blood group and B-CAM antigens are carried by the same molecules.

Close association of the first and fourth extracellular domains of the Duffy antigen/receptor for chemokines by a disulfide bond is required for ligand binding.

It has been demonstrated that the promiscuous chemokine binding profile of the Duffy antigen/receptor for chemokines (DARC) is given by its extracellular NH2-terminal region. However, the relationship among the Fy6, Fya/b, and Fy3 epitopes, localized in the first and fourth extracellular domains of DARC, respectively, and the chemokine binding sites remained a matter of controversy. Here, we performed cross-displacement and cross-inhibition experiments indicating that all anti-Fy6, anti-Fya, and anti-Fy3 monoclonal antibodies and interleukin 8 are antagonists for binding to red cells. Biopanning of phage peptide libraries with an anti-Fy6 monoclonal antibody led to the identification of the motif Phe22-Glu23, the mutation of which altered the binding of both anti-Fy6 and chemokines (interleukin 8, MGSA, RANTES (regulated on activation normal T cell expressed)) to DARC transfectants. These results characterized the core of the Fy6 epitope and provided definitive proof of the tight relationship between Fy6 and the chemokine receptor site. Analysis of red cells treated by sulfhydryl group-modifying reagents suggested that the chemokine receptor function of DARC required the integrity of disulfide bond(s) but not that of free sulfhydryl group(s). Accordingly, mutation of cysteines 51 and 276 abolished chemokine binding to DARC transfectants. Altogether, our results suggested that the chemokine binding pocket of DARC included sequences located in the first and fourth extracellular domains which are brought into close vicinity by a disulfide bridge.

Immunochemical analysis of the Kx protein from human red cells of different Kell phenotypes using antibodies raised against synthetic peptides.

The Kx protein is an erythrocyte membrane polypeptide which is deficient in rare individuals suffering from the McLeod syndrome. The gene encoding this protein has been recently cloned and the Kx protein independently purified as a covalent complex with the Kell blood group protein. To further study the Kx membrane protein, antisera raised in rabbits against six synthetic peptides derived from the primary sequence of this protein were characterized. All antisera but two precipitated the recombinant Kx protein synthesized in coupled transcription-translation in vitro. Three antisera reacted on immunoblots with the 37 kD Kx protein present in the purified Kell-Kx complex and in SDS red cell membrane lysates from variants with different Kell blood group phenotypes, including Ko, which lack the Kell protein of 93 kD. However, no reactivity was found with McLeod preparations lacking Kx protein, thus clearly indicating that these antibodies have a Kx specificity. Unexpectedly, the relative amount of Kx protein in Ko cells was found to be lower than in red cells with common Kell phenotypes, suggesting that the absence of the Kell protein may alter the amount of Kx in the membrane.