Duffy Antigen System - Molecular Biology

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 erythrocytes it is also found on some epithelial cells, 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. In some people who lack the Duffy antigen on their erythrocytes it may still be expressed in some cells. 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 chemotatic 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
• 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 leds to apical retention of intact chemokines and more leukocyte migration.

Binding melanoma growth-stimulating activity inhibits the binding of P. knowlesi to DARC.