Duffy antigen/chemokine receptor (DARC), also known as Fy glycoprotein (FY) or CD234 (Cluster of Differentiation 234), is a protein that in humans is encoded by the ACKR1 gene.
The Duffy antigen is located on the surface of red blood cells, and is named after the patient in whom it was discovered. The protein encoded by this gene is a glycosylated membrane protein and a non-specific receptor for several chemokines. The protein is also the receptor for the human malarial parasites Plasmodium vivax, Plasmodium knowlesi and simian malarial parasite Plasmodium cynomolgi. Polymorphisms in this gene are the basis of the Duffy blood group system.
History
It was noted in the 1920s that black Africans had some intrinsic resistance to malaria, but the basis for this remained unknown. The Duffy antigen gene was the fourth gene associated with the resistance after the genes responsible for sickle cell anaemia, thalassemia and glucose-6-phosphate dehydrogenase.
In 1950, the Duffy antigen was discovered in a multiply-transfused hemophiliac named Richard Duffy, whose serum contained the first example of anti-Fya antibody. In 1951, the antibody to a second antigen, Fyb, was discovered in serum. Using these two antibodies, three common phenotypes were defined: Fy(a+b+), Fy(a+b-), and Fy(a-b+).
Several other types were later discovered bringing the current total up to 5: Fya, Fyb, Fy3, Fy5 and Fy6. Only Fya, Fyb and Fy3 are considered clinically important. Reactions to Fy5 and Fy6 have only rarely been reported. The Fy4 antigen, originally described on Fy (a–b–) RBCs, was obsoleted in 2010 because of scant evidence.
Genetics and genomics
The Duffy antigen/chemokine receptor gene (gp-Fy; CD234) is located on the long arm of chromosome 1 (1.q22-1.q23) and was cloned in 1993. The gene was first localised to chromosome 1 in 1968, and was the first blood system antigen to be localised. It is a single copy gene spanning over 1500 bases and is in two exons. The gene encodes a 336 amino acid acidic glycoprotein. It carries the antigenic determinants of the Duffy blood group system which consist of four codominant alleles—FY*A and FY*B—coding for the Fy-a and Fy-b antigens respectively, FY*X and FY*Fy, five phenotypes (Fy-a, Fy-b, Fy-o, Fy-x and Fy-y) and five antigens. Fy-x is a form of Fy-b where the Fy-b gene is poorly expressed. Fy-x is also known as Fy-bweak or Fy-bWk.
This gene has been redesignated ACKR1.
Fy-a and Fy-b differ by in a single amino acid at position 42: glycine in Fy-a and aspartic acid in Fy-b (guanine in Fy-a and adenosine in Fy-b at position 125). A second mutation causing a Duffy negative phenotype is known: the responsible mutation is G -> A at position 298. The genetic basis for the Fy(a-b-) phenotype is a point mutation in the erythroid specific promoter (a T -> C mutation at position -33 in the GATA box). This mutation occurs in the Fy-b allele and has been designated Fy-bEs (erythroid silent). Two isotypes have been identified. The Fy-x allele is characterized by a weak anti-Fy-b reaction and appears to be the result of two separate transitions: Cytosine265Threonine (Arginine89Cysteine) and Guanine298Adenosine (Alanine100Threonine). A third mutation (a transversion) in this gene has also been described - G145T (Alanine49Serine) - that has been associated with the Fy-x phenotype.
Most Duffy negative black people carry a silent Fy-b allele with a single T to C substitution at nucleotide -33, impairing the promoter activity in erythroid cells by disrupting a binding site for the GATA1 erythroid transcription factor. The gene is still transcribed in non erythroid cells in the presence of this mutation.
The Duffy negative phenotype occurs at low frequency among whites (~3.5%) and is due to a third mutation that results in an unstable protein (Arg89Cys: cytosine -> thymidine at position 265).
The silent allele has evolved at least twice in the black population of Africa and evidence for selection for this allele has been found. The selection pressure involved here appears to be more complex than many text books might suggest. An independent evolution of this phenotype that occurred in Papua New Guinea has also been documented.
A comparative study of this gene in seven mammalian species revealed significant differences between species. The species examined included Pan troglodytes (chimpanzee), Macaca mulatta (rhesus monkey), Pongo pygmaeus (orangutan), Rattus norvegicus (brown rat), Mus musculus (mouse), Monodelphis domestica (opossum), Bos taurus (cow) and Canis familiaris (dog).
Three exons are present in humans and chimpanzees, whereas only two exons occur in the other species. This additional exon is located at the 5' end and is entirely non coding. Both intron and exon size vary considerably between the species examined. Between the chimpanzee and the human, 24 differences in the nucleotide sequence were noted. Of these 18 occurred in non coding regions. Of the remaining 6, 3 were synonymous and 3 non synonymous mutations. The significance of these mutations if any is not known.
The mouse ortholog has been cloned and exhibits 63% homology to the human gene at the amino acid level. The mouse gene is located on chromosome 1 between the genetic markers Xmv41 and D1Mit166. The mouse gene has two exons (100 and 1064 nucleotides in length), separated by a 461 base pair intron. In the mouse DARC is expressed during embryonic development between days 9.5 and 12.
In yellow baboons (Papio cynocephalus) mutations in this gene have been associated with protection from infection with species of the genus Hepatocystis.
The ancestral form of extant DARC alleles in humans appears to be the FY*B allele.
The gene appears to be under strong purifying selection. The cause of this selective pressure has not yet been identified.
Molecular biology
Biochemical analysis of the Duffy antigen has shown that it has a high content of α-helical secondary structure - typical of chemokine receptors. Its N-glycans are mostly of the triantennary complex type terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core.
The Duffy antigen is expressed in greater quantities on reticulocytes than on mature erythrocytes. While the Duffy antigen is expressed on bone marrow erythroblasts and circulating erythrocytes it is also found on Purkinje cells of the cerebellum, endothelial cells of thyroid capillaries, the post-capillary venules of some organs including the spleen, liver and kidney and the large pulmonary venules. Duffy antigen has then a unique cell expression profile in cerebellar neurons, venular endothelial cells and erythroid cells. In some people who lack the Duffy antigen on their erythrocytes it is still expressed in the other cell types.
It has two potential N-linked glycosylation sites at asparagine (Asn) 16 and Asn27.
The Duffy antigen has been found to act as a multispecific receptor for chemokines of both the C-C and C-X-C families, including: monocyte chemotactic protein-1 (MCP-1) - CCL2 regulated upon activation normal T expressed and secreted (RANTES) - CCL5 melanoma growth stimulatory activity (MSGA-α), KC, neutrophil-activating protein 3 (NAP-3) - CXCL1/CXCL2
and the angiogenic CXC chemokines: Growth related gene alpha (GRO-α) - CXCL1 Platelet factor 4 - CXCL4 ENA-78 - CXCL5 Neutrophil activating peptide-2 (NAP-2) - CXCL7 Interleukin-8 (IL-8) - CXCL8
Consequently, the Fy protein is also known as DARC (Duffy Antigen Receptor for Chemokines). The chemokine binding site on the receptor appears to be localised to the amino terminus. The antigen is predicted to have 7 transmembrane domains, an exocellular N-terminal domain and an endocellular C-terminal domain. Alignment with other seven transmembrane G-protein-coupled receptors shows that DARC lacks the highly conserved DRY motif in the second intracellular loop of the protein that is known to be associated with G-protein signaling. Consistent with this finding ligand binding by DARC does not induce G-protein coupled signal transduction nor a Ca2+ flux unlike other chemokine receptors. Based on these alignments the Duffy antigen is considered to be most similar to the interleukin-8B receptors.
Scatchard analysis of competition binding studies has shown high affinity binding to the Duffy antigen with dissociation constants (KD) binding values of 24 ± 4.9, 20 ± 4.7, 41.9 ± 12.8, and 33.9 ± 7 nanoMoles for MGSA, interleukin-8, RANTES and monocyte chemotactic peptide-1 respectively.
In DARC-transfected cells, DARC is internalized following ligand binding and this led to the hypothesis that expression of DARC on the surface of erythrocytes, endothelial, neuronal cells and epithelial cells may act as a sponge and provide a mechanism by which inflammatory chemokines may be removed from circulation as well as their concentration modified in the local environment. This hypothesis has also been questioned after knock out mice were created. These animals appeared healthy and had normal responses to infection. While the function of the Duffy antigen remains presently (2006) unknown, evidence is accumulating that suggests a role in neutrophil migration from the blood into the tissues and in modulating the inflammatory response.
The protein is also known to interact with the protein KAI1 (CD82) a surface glycoprotein of leukocytes and may have a role in the control of cancer.
The Duffy antigen has been shown to exist as a constitutive homo-oligomer and that it hetero-oligomerizes with the CC chemokine receptor CCR5 (CD195). The formation of this heterodimer impairs chemotaxis and calcium flux through CCR5, whereas internalization of CCR5 in response to ligand binding remains unchanged.
DARC has been shown to internalise chemokines but does not scavenge them. It mediates chemokine transcytosis, which leads to apical retention of intact chemokines and more leukocyte migration.
Binding melanoma growth-stimulating activity inhibits the binding of P. knowlesi to DARC.
Population genetics
Differences in the racial distribution of the Duffy antigens were discovered in 1954, when it was found that the overwhelming majority of people of African descent had the erythrocyte phenotype Fy(a-b-): 68% in African Americans and 88-100% in African people (including more than 90% of West African people). This phenotype is exceedingly rare in Whites. Because the Duffy antigen is uncommon in those of Black African descent, the presence of this antigen has been used to detect genetic admixture. In a sample of unrelated African Americans (n = 235), Afro-Caribbeans (n = 90) and Colombians (n = 93), the frequency of the -46T (Duffy positive) allele was 21.7%, 12.2% and 74.7% respectively.
Overall the frequencies of Fya and Fyb antigens in Whites are 66% and 83% respectively, in Asians 99% and 18.5% respectively and in blacks 10% and 23% respectively. The frequency of Fy3 is 100% Whites, 99.9% Asians and 32% Blacks. Phenotype frequencies are: Fy(a+b+): 49% Whites, 1% Blacks, 9% Chinese Fy(a-b+): 34% Whites, 22% Blacks, <1% Chinese Fy(a+b-): 17% Whites, 9% Blacks, 91% Chinese
While a possible role in the protection of humans from malaria was suggested, this was only confirmed clinically in 1976. Since then many surveys have been carried out to elucidate the prevalence of Duffy antigen alleles in different populations including: