Chemokines are best known as signalling molecules that attract leukocytes to sites of inflammation, but recent results show they are also important regulators of hematopoiesis during development.

Animals↗

PubMed1996

The domain on the Duffy blood group antigen for binding Plasmodium vivax and P. knowlesi malarial parasites to erythrocytes.

Plasmodium vivax and the related simian malarial parasite P. knowlesi use the Duffy blood group antigen as a receptor to invade human erythrocytes and region II of the parasite ligands for binding to this erythrocyte receptor. Here, we identify the peptide within the Duffy blood group antigen of human and rhesus erythrocytes to which the P. vivax and P. knowlesi ligands bind. Peptides from the NH2-terminal extracellular region of the Duffy antigen were tested for their ability to block the binding of erythrocytes to transfected Cos cells expressing on their surface region II of the Duffy-binding ligands. The binding site on the human Duffy antigen used by both the P. vivax and P. knowlesi ligands maps to a 35-amino acid region. A 34-amino acid peptide from the equivalent region of the rhesus Duffy antigen blocked the binding of P. vivax to human erythrocytes, although the P. vivax ligand expressed on Cos cells does not bind rhesus erythrocytes. The binding of the rhesus peptide, but not the rhesus erythrocyte, to the P. vivax ligand was explained by interference of carbohydrate with the binding process. Rhesus erythrocytes, treated with N-glycanase, bound specifically to P. vivax region II. Thus, the interaction of P. vivax ligand with human and rhesus erythrocytes appears to be mediated by a peptide-peptide interaction. Glycosylation of the rhesus Duffy antigen appears to block binding of the P. vivax ligand to rhesus erythrocytes.

Amidohydrolases↗

PubMed1996

Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection.

Rare individuals have been multiply exposed to HIV-1 but remain uninfected. The CD4+ T-cells of two of these individuals, designated EU2 and EU3, are highly resistant in vitro to the entry of primary macrophagetropic virus but are readily infectable with transformed T-cell line adapted viruses. We report here on the genetic basis of this resistance. We found that EU2 and EU3 have a homozygous defect in CKR-5, the gene encoding the recently described coreceptor for primary HIV-1 isolates. These individuals appear to have inherited a defective CKR-5 allele that contains an internal 32 base pair deletion. The encoded protein is severely truncated and cannot be detected at the cell surface. Surprisingly, this defect has no obvious phenotype in the affected individuals. Thus, a CKR-5 allele present in the human population appears to protect homozygous individuals from sexual transmission of HIV-1. Heterozygous individuals are quite common (approximately 20%) in some populations. These findings indicate the importance of CKR-5 in HIV-1 transmission and suggest that targeting the HIV-1-CKR-5 interaction may provide a means of preventing or slowing disease progression.

Alleles↗

PubMed1996

The Duffy antigen receptor for chemokines: structural analysis and expression in the brain.

The Duffy antigen receptor for chemokines (DARC) is expressed in human erythrocytes and on endothelial cells lining postcapillary venules in kidney and spleen. DARC is a promiscuous chemokine receptor and a binding protein for the malarial parasite Plasmodium vivax. The expression of DARC by subsets of endothelial cells and neurons in discrete anatomic sites in the brain suggests that this enigmatic receptor may have multiple roles in normal and pathological physiology. Conservation of this promiscuous chemokine binding function is evident from the similarity in nucleotide sequence of DARC homologues from multiple species, as well as the high-affinity binding of human chemokines to murine and avian erythrocytes. Analysis of the functional domains of DARC using chimeric receptors and and monoclonal antibodies to multiple extracellular domains localized chemokine binding to structures in the amino terminal extracellular domain (E1). Scatchard analysis demonstrated that a chimeric DARC receptor, composed of the E1 domain of DARC and the predicted hydrophobic helices and loops of interleukin-8RB (IL-8RB), bound IL-8, and MGSA with KD values almost identical to the wild type receptors and bound a repertoire of C-X-C and C-C chemokines characteristic of DARC. Although numerous reports have demonstrated that chemokines such as IL-8 are expressed in the brain, presumably by glial cells, little insight into the nature of their role in normal or pathological physiology in the nervous system has developed because the target cells that express the corresponding receptors have not yet been identified. Northern blotting experiments suggest that mRNA encoding DARC are expressed in the central nervous system, however, interpretation of this is unclear because of the ubiquitous expression of DARC lining postcapillary venules. This study provides direct evidence to localize expression of DARC in the central nervous system. Immunohistochemical examination of human archival sections of the brain with monoclonal antibodies specific for DARC localize expression of DARC to cell bodies and processes of Purkinjie cells in the cerebellum. The immunohistochemical findings were supported by analysis of chemokine binding and radioligand crosslinking with membranes made from various brain fractions. The hierarchical expression of DARC in neurons in the cerebellum suggest that chemokines may play an important role in the modulation of neuronal activity by glial cells.

Animals↗

PubMed1995

The promiscuous chemokine binding profile of the Duffy antigen/receptor for chemokines is primarily localized to sequences in the amino-terminal domain.

The Duffy antigen (DARC) is a promiscuous chemokine receptor that also binds Plasmodium vivax. DARC belongs to a family of heptahelical chemokine receptors that includes specific (IL-8RA) and shared (IL-8RB) IL-8 receptors. Ligand binding specificity of IL-8 receptors was localized to the amino-terminal extracellular (E1) domain. To determine the basis for promiscuous chemokine binding by DARC, a chimeric receptor composed of the E1 domain of DARC and hydrophobic helices and loops from IL-8RB (DARCe1/IL-8RB) was constructed. Scatchard analysis of stable transfectants demonstrated that the DARCe1/IL-8RB chimeric receptor bound IL-8 and melanoma growth stimulating activity (MGSA) with KD values almost identical to the native receptors. The hybrid receptor also bound RANTES, MCP-1, and MGSA-E6A (which binds DARC, but not IL-8RB), but not MIP-1 alpha, similarly to DARC. Ligand binding to DARC transfectants was unaltered by anti-Fy3, but inhibited by Fy6, which binds an epitope in the E1 domain. The epitope recognized by Fy3 was localized to the third extracellular loop by analysis of insect cells expressing chimeric receptors composed of complementary portions of DARC and IL-8RB. These findings implicate the E1 domain of DARC in multispecific chemokine binding.

Amino Acid Sequence↗

PubMed1995

A mutant of melanoma growth stimulating activity does not activate neutrophils but blocks erythrocyte invasion by malaria.

Alanine scanning mutagenesis of the charged amino acids of melanoma growth stimulating activity (MGSA) was used to identify specific residues that are involved in binding to the human erythrocyte Duffy antigen/chemokine receptor (DARC) and to the type B interleukin-8 receptor (IL-8RB) on neutrophils. Receptor binding and biological studies with the alanine scan mutants of MGSA demonstrate that MGSA binds to DARC and the IL-8RB through distinct binding regions. One of the MGSA mutants, E6A, binds to human erythrocytes and is able to inhibit malaria invasion as efficiently as wild type MGSA but has a severely reduced ability to bind to or signal through the IL-8RB. Mutant chemokines like E6A could prove to be useful therapeutically for the design of receptor blocking drugs that inhibit erythrocyte invasion by Plasmodium vivax malaria.

Alanine↗

PubMed1995

The Duffy antigen/receptor for chemokines (DARC) is expressed in endothelial cells of Duffy negative individuals who lack the erythrocyte receptor.

The Duffy antigen/receptor for chemokines (DARC), first identified on erythrocytes, functions not only as a promiscuous chemokine receptor but also as a receptor for the malarial parasite, Plasmodium vivax. The recent finding that DARC is ubiquitously expressed by endothelial cells lining postcapillary venules provides a possible insight into the function of this receptor because this anatomic site is an active interface for leukocyte trafficking. However, the biological significance of DARC is questionable since it has not yet been determined whether individuals lacking the expression of this protein on their erythrocytes (Duffy negative individuals), who are apparently immunologically normal, express the receptor on endothelial cells. However, we report here that DARC is indeed expressed in endothelial cells lining postcapillary venules and splenic sinusoids in individuals who lack the erythrocyte receptor. These findings are based on immunohistochemical, biochemical, and molecular biological analysis of tissues from Duffy negative individuals. We also present data showing that, in contrast to erythrocyte DARC, cells transfected with DARC internalize radiolabeled ligand. We conclude that the DARC may play a critical role in mediating the effects of proinflammatory chemokines on the interactions between leukocyte and endothelial cells since the molecular pathology of the Duffy negative genotype maintains expression on the latter cell type.

Amino Acid Sequence↗

PubMed1994

The solution structure of melanoma growth stimulating activity.

The solution structure of melanoma growth stimulating activity (MGSA), a dimeric chemokine consisting of 73 residues per monomer, has been determined using two-dimensional homonuclear and three-dimensional heteronuclear NMR spectroscopy. Structure calculations were carried out using a hybrid distance geometry-simulated annealing approach with the programs DGII and X-PLOR. The structure is based on a total of 2362 experimental restraints, comprising 2150 NOE-derived distance restraints (2076 unambiguous intrasubunit restraints, 60 unambiguous intersubunit restraints, and 14 ambiguous restraints with potential contributions from both intra- and intersubunit NOEs), 84 distance restraints for 42 backbone hydrogen bonds, and 128 torsion angle restraints. The ambiguous distance restraints were treated using a target function which accounts for both intra- and intermolecular contributions to the NOE intensity. A total of 25 structures were calculated, with the backbone (N, C alpha, C) atomic r.m.s. distribution about the mean coordinates for residues 8 to 69 being 0.44(+/- 0.10) A for the dimer and 0.34(+/- 0.07) A for the individual monomers. The N- and C-terminal residues (1 to 7 and 70 to 73, respectively) are disordered. The overall structure of the MGSA dimer is similar to that reported previously for the NMR and X-ray structures of interleukin-8 (IL-8), and consists of a six-stranded antiparallel beta-sheet packed against two C-terminal antiparallel alpha-helices. A best fit superposition of the NMR structure of MGSA on the X-ray and NMR structures of IL-8 yields backbone atomic r.m.s. differences of 0.99 and 1.28 A, respectively for individual monomers, and 1.08 and 1.82 A, respectively for the dimers (using MGSA residues 8 to 14 and 19 to 69). In general, the MGSA structure resembles the IL-8 X-ray structure more than it does the IL-8 NMR structure. At the tertiary (monomer) level the two main differences between the MGSA solution structure and IL-8 NMR structure involve the loops between residues 14 to 19 and between residues 30 to 38. At the quaternary (dimer) level the difference results from differing angles between the beta-strands which form the dimer interface, and is manifest as a different interhelical separation (distance of closest approach between the two helices is 15.3 A in the IL-8 NMR structure and 11.7 (+/- 0.4) A in the MGSA structure).

Amino Acids↗

PubMed1994

Identification and characterization of a promiscuous chemokine-binding protein in a human erythroleukemic cell line.

The erythrocyte chemokine receptor is a cell surface protein that binds a wide array of chemokines including interleukin-8 (IL-8), melanoma growth stimulating activity (MGSA), monocyte chemotactic protein-1 (MCP-1), and RANTES (Regulated on Activation, Normal T Expressed and Secreted). This protein has also been identified as the Duffy blood group antigen, a cell surface receptor for the malarial parasite Plasmodium vivax. In the present study, we have identified a chemokine receptor-like binding protein in a human erythroleukemic cell line (HEL), which, based on its molecular properties, may be related to the erythrocyte chemokine receptor. Saturation binding studies with 125I-IL-8 revealed a single class of IL-8 binding sites in HEL cells with a KD of 7.4 +/- 1.9 nM and a receptor density of 12,818 +/- 965 binding sites/cell. In competition studies unlabeled IL-8 MGSA, MCP-1, and RANTES were fully able to inhibit the binding of 125I-IL-8 to HEL cells. Chemical cross-linking with radiolabeled IL-8 resulted in a cross-linked species of 60 kDa in membranes from HEL cells. The labeling was specific since it was inhibited by pre-incubation with 1 microM unlabeled IL-8 or MGSA. A monoclonal antibody (Fy6) to the human erythrocyte Duffy blood group antigen/chemokine receptor blocked the binding of IL-8 and other chemokines to the HEL cell chemokine receptor-like binding protein. Cell membranes from HEL cells and from erythrocyte ghosts were subjected to SDS-PAGE and analyzed by Western blotting with anti-Fy6. The antibody bound to a molecule with a molecular mass of 50 kDa in HEL cell membranes and 40 kDa in erythrocyte ghosts. Northern blot analysis of mRNA revealed that the HEL chemokine-binding protein hybridized to a cDNA probe to the Duffy antigen/chemokine receptor.

Binding Sites↗

PubMed1994

Expression of the Duffy antigen in K562 cells. Evidence that it is the human erythrocyte chemokine receptor.

The human malarial parasite Plasmodium vivax invades erythrocytes by binding to a cell surface protein identified as the Duffy blood group antigen. The molecular properties of the Duffy antigen, which was recently cloned, are very similar to those of a chemokine binding protein known as the human erythrocyte chemokine receptor. This has led to the suggestion that these two molecules are the same protein. To further investigate the suspected double identity of the Duffy antigen we have transfected it into a human erythroleukemic cell line, K562. Cells stably expressing the Duffy antigen were isolated and used to characterize the protein. K562 cells transfected with the Duffy antigen displayed specific 125I-melanoma growth-stimulating activity (MGSA) binding while mock transfected cells did not. Comparison of 125I-MGSA binding to the Duffy antigen and the human erythrocyte chemokine receptor showed that the specific 125I-MGSA binding to both proteins was displaced by excess unlabeled MGSA, interleukin-8, RANTES, monocyte chemotactic peptide-1, and platelet factor 4, but not by macrophage inflammatory protein-1 alpha or -1 beta. Scatchard analysis of competition binding studies with these unlabeled chemokines revealed high affinity binding to the Duffy antigen with KD binding values of 24 +/- 4.9, 20 +/- 4.7, 41.9 +/- 12.8, and 33.9 +/- 7 nM for MGSA, interleukin-8, RANTES, and monocyte chemotactic peptide-1, respectively. A monoclonal antibody, Fy6, to the Duffy antigen inhibited 125I-MGSA binding to K562 cells expressing the Duffy antigen. Cell membranes from K562 cells permanently expressing the Duffy antigen were chemically cross-linked with 125I-MGSA. SDS-polyacrylamide gel electrophoresis analysis of the cross-linked products showed covalent incorporation of radiolabeled MGSA into a protein of molecular mass 47 kDa, and cross-linking was inhibited in the presence of unlabeled MGSA. These studies provide evidence that the Duffy blood group antigen is the same protein as the human erythrocyte chemokine receptor.

Amino Acid Sequence↗

PubMed1994

Molecular properties of the chemokine receptor family.

Chemokine receptors play a major role in the mobilization and activation of the cells of the immune system, and are discussed in this review by Richard Horuk. Six receptors that bind chemokines with high affinity have been cloned and all belong to the superfamily of G protein-coupled receptors. Chemokine receptors are mainly expressed in immune cells, which are their major target cells. However, two of the cloned receptors are expressed by viruses and may play a role in protecting the virus from immune surveillance. In addition, a novel chemokine receptor that is expressed in human erythrocytes not only binds CC and CXC chemokines with high affinity but is also a receptor for the malarial parasite Plasmodium vivax.

Amino Acid Sequence↗

PubMed1994

The interleukin-8-receptor family: from chemokines to malaria.

The interleukin 8 (IL-8)-receptor family includes two specific receptors (type A and B) that both bind IL-8 with high affinity. These receptors have been cloned, and belong to a superfamily of G-protein-linked receptors that signal in response to IL-8 on a variety of cell types. In contrast to these receptors, which have a narrow ligand-binding profile, a promiscuous IL-8 receptor has been found on human erythrocytes that binds a variety of chemokines with high affinity. This protein, known as the chemokine receptor, was recently shown to bind the malarial parasite Plasmodium vivax, and may play a major role in inflammation by limiting the concentration of soluble chemokines in the circulation.

Amino Acid Sequence↗

PubMed1994

Postcapillary venule endothelial cells in kidney express a multispecific chemokine receptor that is structurally and functionally identical to the erythroid isoform, which is the Duffy blood group antigen.

The human erythrocyte chemokine receptor has recently been shown to be identical to the Duffy blood group antigen and is expressed in multiple organs, including kidney. Here we have examined the molecular properties of the renal isoform. Immunoblot analysis of erythrocyte and kidney detergent lysates, with a monoclonal antibody (Fy6) to the Duffy antigen, revealed that the renal isoform had a molecular mass of 43-45 kD, which could be distinguished from that observed in erythroid cells (38-47 kD). Chemical cross-linking of kidney membranes to 125I-melanoma growth stimulatory activity (MGSA) indicated that the renal chemokine receptor had a molecular mass of 38-45 kD. Binding of 125I-labeled MGSA to kidney membranes was competitively inhibited by the addition of unlabeled MGSA, IL-8, regulated on activation, normal T expressed and secrted, and monocyte chemotactic protein-1. Scatchard analysis of MGSA binding showed that the chemokine receptor from renal tissues had a binding affinity of 3.5 nM similar to that observed for the erythroid isoform (5-10 nM). The primary structure of the renal chemokine receptor predicted from the nucleotide sequence of cDNA from renal tissues is identical to that reported for the erythroid isoform. Immunocytochemical staining of kidney with Fy6 localized expression to endothelial cells present in postcapillary venules. These studies implicate the Duffy antigen/chemokine receptor in the complex interactions between postcapillary endothelial cells and granulocytes, which are modulated by pro-inflammatory chemokines.

Antibodies, Monoclonal↗