Christophe Tournamille

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 susbstitution (G131A) changing Gly to Asp at position 44 of the gpD protein. When expressed in simian Cos-7 cells, the Fy(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.

Relative immunogenicity of Fya and K antigens in a Caucasian population, based on HLA class II restriction analysis.

BACKGROUND: It has long been known that relative immunogenicity is a characteristic of protein red blood cell (RBC) antigens, but the mechanisms remain unclear. The aim of this work was to elucidate the mechanisms underlying this relative immunogenicity.

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 Duffyantigen/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.

Partial C antigen in sickle cell disease patients: clinical relevance and prevention of alloimmunization

BACKGROUND: Partial Rh antigens have been widely described in black individuals. Carriers are prone to immunization when exposed to the normal antigens. In sickle cell disease (SCD), patient alloimmunization is a major cause of transfusion failure. The potential of individuals with partial C antigen to make anti‐C has not been investigated. We sought partial C status and anti‐C production in a cohort of SCD patients with the C+ phenotype, to determine whether exposure to normal C antigen should be avoided.

Structure-function analysis of the extracellular domains of the Duffy antigen/receptor for chemokines: characterization of antibody and chemokine binding sites.

Summary. The Duffy antigen/receptor for chemokines (DARC), a seven‐transmembrane glycoprotein carrying the Duffy (Fy) blood group, acts as a widely expressed promiscuous chemokine receptor. In a structure–function study, we analysed the binding of chemokines and anti‐Fy monoclonal antibodies (mAbs) to K562 cells expressing 39 mutant forms of DARC with alanine substitutions spread out on the four extracellular domains (ECDs). Using synthetic peptides, we defined previously the Fy6 epitope (22‐FEDVW‐26), and we characterized the Fya epitope as the linear sequence 41‐YGANLE‐46. In agreement with these results, mutations of F22‐E23, V25 and Y41, G42, N44, L45 on ECD1 abolished the binding of anti‐Fy6 and anti‐Fya mAbs to K562 cells respectively, Anti‐Fy3 binding was abolished by D58–D59 (ECD1), R124 (ECD2), D263 and D283 (ECD4) substitutions. Mutations of C51 (ECD1), C129 (ECD2), C195 (ECD3) and C276 (ECD4 severely reduced anti‐Fy3 and CXC‐chemokine ligand 8 (CXCL‐8) binding. CXCL‐8 binding was also abrogated by mutations of F22–E23, P50 (ECD1) and D263, R267, D283 (ECD4). These results defined the Fya epitope and suggested that (1) two disulphide bridges are involved in the creation of an active chemokine binding pocket; (2) a limited number of amino acids in ECDs 1–4 participate in CXCL‐8 binding; and (3) Fy3 is a conformation‐dependent epitope involving all ECDs. We also showed that N‐glycosylation of DARC occurred on N16SS and did not influence antibody and chemokine binding.

Sequence, evolution and ligand binding properties of mammalian Duffy antigen/receptor for chemokines

The Duffy antigen/receptor for chemokine, DARC, acts as a widely expressed promiscuous chemokine receptor and as the erythrocyte receptor for Plasmodium vivax. To gain insight into the evolution and structure/function relations of DARC, we analyzed the binding of anti-human Fy monoclonal antibodies (mAbs) and human chemokines to red blood cells (RBCs) from 11 nonhuman primates and two nonprimate mammals, and we elucidated the structures of the DARC genes from gorilla, gibbon, baboon, marmoset, tamarin, night monkey and cattle. CXCL-8 and CCL-5 chemokine binding analysis indicated that the promiscuous binding profile characteristic of DARC is conserved across species. Among three mAbs that detected the Fy6 epitope by flow cytometric analysis of human and chimpanzee RBCs, only one reacted with night monkey and squirrel monkey. Only chimpanzee RBCs bound a significant amount of the anti-Fy3 mAb. Fy3 was also poorly detected on RBCs from gorilla, baboon and rhesus monkey, but not from new world monkeys. Alignment of DARC homologous sequences allowed us to construct a phylogenetic tree in which all branchings were in accordance with current knowledge of primate phylogeny. Although DARC was expected to be under strong internal and external selection pressure, in order to maintain chemokine binding and avoid Plasmodium vivax binding, respectively, our present study did not provide arguments in favor of a selection pressure on the extracellular domains involved in ligand specificity. The amino acid variability of DARC-like polypeptides was found to be well correlated with the hydrophylicity indexes, with the highest divergence on the amino-terminal extracellular domain. Analysis of the deduced amino acid sequences highlighted the conservation of some amino acid residues, which should prove to be critical for the structural and functional properties of DARC.

Red blood cell immunization in sickle cell disease: evidence of a large responder group and a low rate of anti-Rh linked to partial Rh phenotype

The main side-effect of transfusion is alloimunization against red blood cell (RBC) antigens. Thirteen percent of the general population were shown to be responders and 30% of responders make antibodies indicating a rate of alloimmunization of 3.9%.[1][1] However, alloimmunization is more frequent

Relevance of RH variants in transfusion of sickle cell patients

Transfusion remains the main treatment of sickle cell disease patients. Red cell alloimmunization is frequent because of the antigen disparities between patients of African descent and donors of European ancestry. Alloimmunization is associated with severe hemolytic transfusion reaction, autoantibody formation, and difficulties in the management of transfusion compatibility. Beside common antigens, a number of different RH variant antigens found in individuals of African descent can be involved in alloimmunization. If some variants, such as Hr(S) negative antigens, are known to prone significant alloantibodies and delayed hemolytic transfusion reactions, it is not clear whether all the described variants represent a clinical risk for sickle cell disease patients. The knowledge of the clinical relevance of RH variants is a real issue. An abundance of molecular tools are developed to detect variants, but they do not distinguish those likely to prone immunization from those that are unlikely to prone immunization and delayed hemolytic transfusion reactions. A strategy of prevention, which generally requires rare red blood cells, cannot be implemented without this fundamental information. In this review, we discuss the relevance of RH variants in sickle cell disease, based on the published data and on our experience in transfusion of these patients.

Weak D phenotypes and transfusion safety: where do we stand in daily practice?

BACKGROUND: Weak D Types 1, 2, and 3 recipients cannot be immunized when exposed to D antigen. Molecular biology is very efficient to type weak D variants but rarely implemented in daily practice. The serologic typing practice of weak D in a Caucasian patient population was analyzed and a transfusion strategy is proposed.

Bases moléculaires et relations structure-fonction des antigènes de groupe sanguin Duffy: récepteur de chimiokines et de Plasmodium vivax

Duffy blood group antigens are of major interest in clinical medicine as they are not only involved in blood transfusion risks and occasionally in neonatal hemolytic disease, but also in the invasion of red blood cells by the hemoparasitic Plasmodium vivax. The FY locus maps to chromosome 1q22-q23, and is composed of 4 alleles: FY*A and FY*B (coding for the Fya and Fyb antigens, respectively), FY*X and FY*Fy. The Duffy antigens are carried by a 336 amino-acid glycoprotein named the Duffy Antigen/Receptor for Chemokines (DARC) that can bind with high affinity selected members of the CXC and CC classes of chemokines. Today, the genetic bases of the Duffy system have been characterized. The identification of the polymorphisms associated with the 4 alleles FY*A, FY*B, FY*Fy and FY*X has led to the development of a complete genotyping of the Duffy system by PCR, which increases the safety and lessens the risk of blood transfusion, and is useful in determining feto-maternal incompatibilities and in genetic filiation analyses. DARC is not solely expressed in erythroid cells: the same polypeptide isoform is found on the surface of endothelial cells of post-capillary venules throughout the body and also on the surface of Purkinje cells in the cerebellum, although it is encoded by different RNA messengers in each case, i.e., 1.35 and 7.5 kb, respectively. The preliminary analyses of receptor-ligand interaction have shown the existence of a chemokine-binding pocket defined by the close proximity of the first and fourth transmembrane domains of the DARC protein, and also by the importance of the N-terminal extracellular region for the binding of Plasmodium vivax merozoites.