The Duffy protein: a malarial and chemokine receptor.

A major advance towards understanding the Duffy blood group system has been achieved with the cloning of FY, a single-copy gene located in the 1q22->q23 region of chromosome 1. The product of FY Is an acidic glycoprotein (gp-Fy), which spans the plasma membrane seven times and has an exocellular N-terminal domain and an endocellular C-terminal domain. The system consists of four alleles, five phenotypes, and five antigens. FYA, FYB, FYB(ES), and FYB(WK) are the alleles; Fy(a+b-), Fy(a-b+), Fy(a+b+), Fy(a-b+(wK)), and Fy(a-b-), are the phenotypes, and Fy(a), Fy(b), Fy3, Fy5, and Fy6 are the antigens. Fy(a-b-), or Duffy-negative individuals, lack the Duffy protein on erythrocytes and are predominantly African and American blacks. They have the FYB(Es) allele with a mutation in the promoter region, which abolishes the expression of the protein in erythrocytes only. In the few cases of non-black Fy(a-b-) individuals, a nonsense mutation prevents the synthesis of gp-Fy. In Fy(a-b+(wk)) erythrocytes, the Fy(b) antigen is weakly expressed due to a reduced amount of the protein. The Fy5 antigen includes the Rh protein, and the Fy6 antigen is defined by a murine monoclonal antibody. Gp-Fy is produced in several cell types, including endothellal cells of capillary and postcapillary venules, epithelial cells of kidney collecting ducts, and lung alveoli, as well as PurkinJe cells of the cerebellum. The Duffy protein plays a role in inflammation and in malaria Infection. The protein is a member of the superfamily of chemokine receptors and is the receptor to which certain malarial parasites bind to invade red blood cells. The parasite-specific binding site, the binding site of chemokines, and the major antigenic domains are located in overlapping regions at the exocellular N terminus of the Duffy protein.

Deletion of the murine Duffy gene (Dfy) reveals that the Duffy receptor is functionally redundant.

All of the antigenic determinants of the Duffy blood group system are in a glycoprotein (gp-Fy), which is encoded by a single-copy gene (FY) located on chromosome 1. gp-Fy is also produced in several cell types, including endothelial cells of capillary and postcapillary venules, the epithelial cell of kidney collecting ducts, lung alveoli, and the Purkinje cells of the cerebellum. This protein, which spans the cell membrane seven times, is a member of the superfamily of chemokine receptors and a malarial parasite receptor. The mouse Duffy gene (Dfy) homolog of human FY is also a single-copy gene, which maps in a region of conserved synteny with FY and produces a glycoprotein with 60% homology to the human protein. The mouse Duffy-like protein also binds chemokines. To study the biological role of gp-Fy, we generated a mouse strain in which Dfy was deleted. These homozygous Dfy(-/-) mice were indistinguishable in size, development, and health from wild-type and heterozygous littermates. We also examined components of the immune system and found no differences in lymph nodes or peripheral blood leukocyte levels between knockout and wild-type mice. The gross and histological anatomy of the thymus, spleen, lung, and brain showed no significant differences between mutants and wild-type mice. There was no indication of an overall difference between the knockout and wild-type mice in systematic neurological examinations. The only significant difference between Dfy(-/-) and Dfy(+/+) mice that we found was in neutrophil migration in peritoneal inflammations induced by lipopolysaccharide and thioglycolate. In mice homozygous for the deletion, there was less neutrophil recruitment into the peritoneal cavity and neutrophil influx in the intestines and lungs than in wild-type mice. Despite this, the susceptibility to Staphylococcus aureus infection was the same in the absence and in the presence of gp-Fy. Our results indicate that gp-Fy is functionally a redundant protein that may participate in the neutrophil migratory process.

Cloning, characterization, and mapping of a murine promiscuous chemokine receptor gene: homolog of the human Duffy gene.

We report here the isolation and genomic organization of the orthologous mouse Duffy gene, named Dfy. It is a single copy gene located in chromosome 1 in a region homologous to the human Duffy gene (FY). Sequence analyses indicate that Dfy consists of two exons: exon 1 of 55 nucleotides, which encodes 7 amino acid residues; and exon 2 of 1038 nucleotides, which encodes 327 residues. The single intron consists of 462 nucleotides. The 5'-end promoter region contains motifs involved in vertebrate development in addition to potential binding sites of factors for globin transcription. The open reading frame (ORF) shows 60% homology with the human Duffy protein. However, mouse erythrocytes are serologically Duffy-negative and mouse erythrocyte membrane proteins do not cross-react with two Duffy-specific rabbit polyclonal antibodies. The deduced protein predicts a M(r) of 36,692 and carries three potential N-glycosylation sites to asparagine residues. Hydropathy analysis predicts an exocellular amino-terminal domain of 57 residues, seven transmembrane alpha-helices, and an endocellular carboxy-terminal domain of 29 residues. In bone marrow and spleen, Dfy expresses a major 1.4-kb and a minor 1.8-kb mRNA. Contrary to humans, Dfy is expressed in liver, synthesizing a 1.4-kb mRNA, and is repressed in kidney. Dfy is highly expressed in mouse brain and produces a major 8.5-kb and a minor 10.2-kb mRNA. The human erythroleukemia K562 cells, transfected with cDNA encoding the mouse Duffy-like protein and mouse erythrocytes, have the same chemokine binding profiles indicating that they contain the same protein.

Detection of Duffy antigen in the plasma membranes and caveolae of vascular endothelial and epithelial cells of nonerythroid organs.

The nonerythroid expression of the Duffy blood group protein (gp-Fy) was confined to certain cell types. Immunocytochemistry studies of the kidney showed gp-Fy in the endothelium of glomeruli, peritubular capillaries, vasa recta, and the principal cells (epithelial) of collecting ducts. Gp-Fy was also produced in the endothelial cells of large venules and epithelial cells (type-I) of pulmonary alveoli. In the thyroid, only the endothelial cells of capillaries produced gp-Fy. In the spleen, the endothelial cells of capillaries, high endothelial venule, and sinusoids produced abundant gp-Fy. Ultrastructural studies showed that apical and basolateral plasma membrane domains, including caveolae, had gp-Fy. Immunoblot analysis showed substantially less gp-Fy in nonerythroid cells than in erythrocytes. Moreover, the analyzed nonerythroid organs of Duffy-negative individuals did not produce more gp-Fy to compensate for the lack of this protein in their erythrocytes. The nucleotide sequence and the size of kidney mRNA from a Duffy-positive individual were the same as that of bone marrow. It is assumed, therefore, that nonerythroid Duffy protein is the product of the same gene as that of bone marrow. This notion is reinforced by the fact that nonerythroid and erythroid gp-Fy have the same antigenic domains.

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.

The coding sequence of Duffy blood group gene in humans and simians: restriction fragment length polymorphism, antibody and malarial parasite specificities, and expression in nonerythroid tissues in Duffy-negative individuals.

The coding and untranslated flanking sequences of Duffy gene (FY) in humans and simians are in a single exon. The difference between the two codominant alleles, FY*A and FY*B, is a single change at nucleotide 306: guanidine is in FY*A and adenine is in FY*B. This produces a codon change that subsequently modifies the amino acid at position 43 of gpFy, the major subunit of the Duffy blood group protein complex. The glycine at this position in antigen Fya exchanges with aspartic acid in antigen Fyb. The guanidine at nucleotide 306 creates an additional Ban I restriction site in FY*A. Ban I digestion of DNA-PCR amplified products of FY*B and FY*A yields three and four fragments, respectively. Restriction fragment length polymorphism (RFLP) studies show that Fy(a+b-) and Fy(a-b+) whites are FY homozygous, that most Fy(a-b-) blacks have FY*B, and most Fy(a+b-) blacks are FY*A/FY*B heterozygous. In the black population a silent FY*B is very common, but a silent FY*A has not been found yet. On RNA blot analysis, the gpFy cDNA clone detected mRNA in the lung, spleen, and colon but not in the bone marrow of Duffy-negative individuals. Therefore, there is no null phenotype in Fy(a-b-) blacks. The gpFy homology between human and chimpanzee is 99% with a single residue change at position 116 (valine to isoleucine), whereas a 94% homology is found in squirrel and rhesus monkeys, and there is a 93% homology in aotus monkey when compared with humans. The N-terminal exocellular domain of simian gpFy helps to identify a set of amino acids critical for antibody and malarial parasite specificities.

Duffy and receptors for P. vivax and chemotactic peptides.

The Duffy blood group system consists of two principal antigens, Fya and Fyb produced by FY*A and FY*B co-dominant alleles. Antisera, anti-Fya and anti-Fyb, define four phenotypes: Fy(a+b-), Fy(a-b+), Fy(a+b+) and Fy(a-b-). Neither antiserum agglutinates Fy(a-b-) cells, the predominant phenotype in Blacks. Outside the Black population, Fy(a-b-) phenotype is very rare. Duffy antigens appear to be multimeric erythrocyte-membrane proteins composed of different subunits. A glycoprotein of 35-45 kDa, gp-Fy, is the major subunit of the complex and has antigenic determinants defined by Duffy antibodies. The protein consists of 337 amino acid residues with a M(r) of 35,733, the same as deglycosylated gp-Fy. The hydropathy map predicts an exocellular N-terminal domain of 64 residues, nine transmembrane alpha-helices, three short protruding hydrophilic loops and an endocellular C-terminal domain of 23 residues. Duffy specific transcript, a approximately 1.3 kb mRNA, is produced by the bone marrow of Duffy-positive individuals, but it is not produced by the bone marrow of Duffy-negative individuals. The same size mRNA is produced in many tissues of Duffy-positive individuals. The same tissues of Duffy-negative individuals also synthesize the same size mRNA and the same gp-Fy as that of Duffy-positive individuals. There is not, therefore, Duffy null phenotype in the Black population. The difference between FY*A and FY*B alleles is a single nucleotide change at position 306; guanine is in FY*A, and adenine is in FY*B.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Confirmation of Duffy blood group antigen locus (FY) at 1q22-->q23 by fluorescence in situ hybridization.

The gene for the Duffy blood group antigen (FY) was previously assigned to the chromosome region 1q22-->q23 by linkage analysis. We confirm this localization by fluorescence in situ hybridization.

Cloning of glycoprotein D cDNA, which encodes the major subunit of the Duffy blood group system and the receptor for the Plasmodium vivax malaria parasite.

cDNA clones encoding the major subunit of the Duffy blood group were isolated from a human bone marrow cDNA library using a PCR-amplified DNA fragment encoding an internal peptide sequence of glycoprotein D (gpD) protein. The open reading frame of the 1267-bp cDNA clone indicated that gpD protein was composed of 338 amino acids, predicting a M(r) of 35,733, which was the same as a deglycosylated gpD protein. Portions of the predicted amino acid sequence, matched with six CNBr/pepsin peptides obtained from affinity-purified gpD protein. In ELISA analysis, an anti-Duffy murine monoclonal antibody reacted with a synthetic peptide deduced from the cDNA clone. Hydropathy analysis suggested the presence of 9 membrane-spanning alpha-helices. In bone marrow RNA blot analysis, the gpD cDNA detected a 1.27-kb mRNA in Duffy-positive but not in Duffy-negative individuals. It also identified the same size mRNA in adult kidney, adult spleen, and fetal liver; in brain, it detected a prominent 8.5-kb and a minor 2.2-kb mRNA. In Southern blot analysis, gpD cDNA identified a single gene in Duffy-positive and -negative individuals. Duffy-negative individuals, therefore, have the gpD gene, but it is not expressed in bone marrow. The same or a similar gene is active in adult kidney, adult spleen, and fetal liver of Duffy-positive individuals. Whether this is true in Duffy-negative individuals remains to be demonstrated. A GenBank sequence search yielded a significant protein sequence homology to human and rabbit interleukin-8 receptors.

Purification and characterization of an erythrocyte membrane protein complex carrying Duffy blood group antigenicity. Possible receptor for Plasmodium vivax and Plasmodium knowlesi malaria parasite.

A murine monoclonal antibody, named anti-Fy6, which agglutinates all human red cells except those of Fy(a-b) phenotype was used for purification and characterization of Duffy antigens. Duffy antigens are multimeric red cell membrane proteins composed of different subunits of which only one, designated pD protein, reacts in immunoblots with the murine monoclonal antibody anti-Fy6. Affinity-purified detergent-soluble antigen-antibody complex obtained from red cells, surface-labeled with 125I yielded a complex pattern of bands when separated by polyacrylamide gel electrophoresis. Proteins that react with anti-Fy6 in immunoblots are: pA and pB (greater than 100 kDa) and pD (36-46 kDa). Electroeluted pD protein aggregates and generates bands of similar molecular mass to pA and pB proteins. Electroeluted pA and pB proteins disaggregate yielding pD protein. Oligomers and monomers of pD protein are present in red cells carrying Duffy antigens and absent in Fy(a-b-) cells. Six other proteins of molecular weight ranging from 68 to 21 kDa either associate or co-purify with pD protein. These proteins are only present in Duffy antigen positive cells. The pD protein is different in Fy(a+b-) and Fy(a-b+) cells by fingerprint analysis. Human antisera identify the same proteins in red cell carrying Duffy antigens as the murine monoclonal antibody anti-Fy6.

Localization of the McLeod locus (XK) within Xp21 by deletion analysis.

The McLeod phenotype is an X-linked, recessive disorder in which the red blood cells demonstrate acanthocytic morphology and weakened antigenicity in the Kell blood group system. The phenotype is associated with a reduction of in vivo red cell survival, but the permanent hemolytic state is usually compensated by erythropoietic hyperplasia. The McLeod phenotype is accompanied by either a subclinical myopathy and elevated creatine kinase (CK) or X-linked chronic granulomatous disease (CGD). Seven males with the McLeod red-blood-cell phenotype and associated myopathy but not CGD, one male with the McLeod phenotype associated with CGD, and two males known to possess large deletions of the Duchenne muscular dystrophy (DMD) locus were studied. DNA isolated from each patient was screened for the presence or absence of various cloned sequences located in the Xp21 region of the human X chromosome. Two of the seven males who have only the McLeod phenotype and are cousins exhibit deletions for four Xp21 cloned fragments but are not deleted for any portion of either the CGD or the DMD loci. Comparison of the cloned segments absent from these two McLeod cousins with those absent from the two DMD boys and the CGD/McLeod patient leads to the submapping of various cloned DNA segments within the Xp21 region. The results place the locus for the McLeod phenotype within a 500-kb interval distal from the CGD locus toward the DMD locus.

Interstrand duplexes in Friend erythroleukemia nuclear RNA. The interaction of non-polyadenylated nuclear RNA with polyadenylated nuclear RNA and with small nuclear RNAs.

Intermolecular duplexes among large nuclear RNAs, and between small nuclear RNA and heterogeneous nuclear RNA, were studied after isolation by a procedure that yielded protein-free RNA without the use of phenol or high salt. The bulk of the pulse-labeled RNA had a sedimentation coefficient greater than 45 S. After heating in 50% (v/v) formamide, it sedimented between the 18 S and 28 S regions of the sucrose gradient. Proof of the existence of interstrand duplexes prior to deproteinization was obtained by the introduction of interstrand cross-links using 4'-aminomethyl-4,5',8-trimethylpsoralen and u.v. irradiation. Thermal denaturation did not reduce the sedimentation coefficient of pulse-labeled RNA obtained from nuclei treated with this reagent and u.v. irradiated. Interstrand duplexes were observed among the non-polyadenylated RNA species as well as between polyadenylated and non-polyadenylated RNAs. beta-Globin mRNA but not beta-globin pre-mRNA also contained interstrand duplex regions. In this study, we were able to identify two distinct classes of polyadenylated nuclear RNA, which were differentiated with respect to whether or not they were associated with other RNA molecules. The first class was composed of poly(A)+ molecules that were free of interactions with other RNAs. beta-Globin pre-mRNA belongs to this class. The second class included poly(A)+ molecules that contained interstrand duplexes. beta-Globin mRNA is involved in this kind of interaction. In addition, hybrids between small nuclear RNAs and heterogeneous nuclear RNA were isolated. These hybrids were formed with all the U-rich species, 4.5 S, 4.5 SI and a novel species designated W. Approximately equal numbers of hybrids were formed by species U1a, U1b, U2, U6 and W; however, species U4 and U5 were significantly under-represented. Most of these hybrids were found to be associated stably with non-polyadenylated RNA. These observations demonstrated for the first time that small nuclear RNA-heterogeneous nuclear RNA hybrids can be isolated without crosslinking, and that proteins are not necessary to stabilize the complexes. However, not all molecules of a given small nuclear RNA species are involved in the formation of these hybrids. The distribution of a given small nuclear RNA species between the free and bound state does not reflect the stability of the complex in vitro but rather the abundance of complementary sequences in the heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 400 WORDS)

The stringent and relaxed phenomena in Saccharomyces cerevisiae.

We present here a detailed analysis of the effect of amino acid starvation and the addition of cycloheximide on RNA metabolism of yeast cells and spheroplasts. These effects have been studied at the level of uridine phosphorylation, methylation of rRNA, and biosynthesis of 35 S, 4 S, and 5 S RNA species. Amino acid starvation inhibits the phosphorylation of uridine assigned for RNA synthesis more than that for other metabolic processes. This implies that a salvage pathway for the synthesis of UMP and CMP is regulated by the rate of transcription and perhaps is localized in the nucleus. The rate of rRNA methylation is not coupled with the rate of transcription; therefore, quantitation of 35 S RNA synthesis (yeast rRNA primary transcript by [methyl-3H]methionine labeling is unreliable. Biosynthesis of 35 S RNA ceases immediately after the cells are transferred to an amino-acid-deficient medium; at a later time 4 S and 5 S RNAs are also inhibited. Therefore, coordination and noncoordination in the stringent response of these RNA species depend upon the time of starvation. Although addition of a small dose (less than 1.0 microgram/ml) of cycloheximide to starved yeast spheroplasts does not alter the rate of uridine phosphorylation, it increases the rate of entrance of uridine into total RNA. This effect is of greater magnitude in 4 S and 5 S than in 35 S RNA. Since the drug does not alter the rate of decay of 35 S RNA that takes place in starvation, it has a selective effect on transcription. A similar small dose, however, produces inhibition of transcription of all these RNA species in nonstarved conditions. This opposite effect of the drug appears to be a characteristic feature of RNA metabolism in eukaryotes.

Isolation and characterization of the nuclear matrix in Friend erythroleukemia cells: chromatin and hnRNA interactions with the nuclear matrix.

Nuclear matrices from undifferentiated and differentiated Friend erythroleukemia cells have been obtained by a method which removes DNA in a physiological buffer. These matrices preserved the characteristic topographical distribution of condensed and diffuse "chromatin" regions, as do nuclei in situ or isolated nuclei. Histone H1 was released from the nuclear matrix of undifferentiated cells by 0.3 M KCl; inner core histones were released by 1 M KCl. Nuclear matrix from differentiated cells did not maintain H1, and histone cores were fully released in 0.7 M KCl. KCl removed the core histones as an octameric structure with no evidence of preferential release of any single histone. Electron microscopy of KCl-treated matrix revealed no condensed regions but rather a network of fibrils in the whole DNA-depleted nuclei. When nuclear matrices from both types of cell were exposed to conditions of very low ionic strength, inner core histones and condensed regions remained. These observations support the contention that inner core histones are bound to matrix through natural ionic bonds or saline-labile elements, and that these interactions are implicated in chromatin condensation. hnRNA remained undegraded and tenaciously associated to the matrix fibrils, and was released only by chemical means which, by breaking hydrophobic and hydrogen bonds, produced matrix lysis. Very few nonhistone proteins were released upon complete digestion of DNA from either type of nuclei. The remaining nonhistone proteins represent a large number of species of which the majority may be matrix components. The molecular architecture in both condensed and diffuse regions of interphase nuclei appears to be constructed of two distinct kinds of fibers; the thicker chromatin fibers are interwoven with the thinner matrix fibers. The latter are formed by a heteropolymer of many different proteins.

Cross-linking of proteins in nuclei and DNA-depleted nuclei from Friend erythroleukemia cells.

Reversible cross-linking of proteins in nuclei and DNA-depleted nuclei from Friend erythroleukemia cells was used as a probe to determine whether the protein structure was preserved following treatment with DNAase I. Interactions between histones were analyzed through cross-linking with 2-iminothiolane or dimethyl 3,3'-dithiobispropionimidate. No alterations in the interactions between intranucleosomal histone proteins resulted from digestion of the nuclear DNA. There was, however, a diminished extent of cross-linking of histone H1 to itself and to the intranucleosomal histones in DNA-depleted nuclei. The interactions of a group of nonhistone proteins with histone H3 could be monitored by cross-linking through the formation of disulfide bonds caused by oxidation of nuclei with H2O2. These interactions were not markedly affected by treatment of the nuclei with DNAase I. However, differences were observed in the extent of cross-linking of some of these proteins when cross-linking in nuclei from undifferentiated cells was compared to that in nuclei from cells which had been induced to differentiate with dimethylsulfoxide.