Ultrastructure and Function of the Fractalkine Mucin Domain in CX3C Chemokine Domain Presentation

Abstract

Fractalkine (FKN), a CX 3 C chemokine/mucin hybrid molecule on endothelium, functions as an adhesion molecule to capture and induce firm adhesion of a subset of leukocytes in a selectin-and integrin-independent manner. We hypothesized that the FKN mucin domain may be important for its function in adhesion, and tested the ability of secreted alkaline phosphatase (SEAP) fusion proteins containing the entire extracellular region (FKN-SEAP), the chemokine domain (CX3C-SEAP), or the mucin domain (mucin-SEAP) to support firm adhesion under flow. CX3C-SEAP induced suboptimal firm adhesion of resting peripheral blood mononuclear cells, compared with FKN-SEAP, and mucin-SEAP induced no firm adhesion. CX3C-SEAP and FKN-SEAP bound to CX 3 CR1 with similar affinities. By electron microscopy, fractalkine was 29 nm in length with a long stalk (mucin domain), and a globular head (CX 3 C). To test the function of the mucin domain, a chimeric protein replacing the mucin domain with a rod-like segment of E-selectin was constructed. This chimeric protein gave the same adhesion of peripheral blood mononuclear cells as intact FKN, both when immobilized on glass and when expressed on the cell surface. This implies that the function of the mucin domain is to provide a stalk, extending the chemokine domain away from the endothelial cell surface to present it to flowing leukocytes.Leukocyte trafficking of cells out of the bloodstream and into sites of inflammation requires multiple steps (1, 2). In the classic pathway of leukocyte migration, the first step involves transient, selectin-mediated interactions between the rolling leukocytes and the endothelium (3, 4). In the next step, integrins are activated by locally produced chemokines to induce firm adhesion of the leukocyte to endothelial cells (5-7). Leukocytes then extravasate through the vascular wall and into the tissue.Recently, a new pathway by which leukocytes can be induced to firmly adhere to endothelium and traffic into inflamed tissues has been described. This process is mediated by the chemokine fractalkine (FKN), 1 expressed on endothelial cells, and its G-protein coupled receptor, CX 3 CR1, expressed on PBMC. Fractalkine (neurotactin in the mouse) is the first, and thus far only, member of the CX 3 C chemokine family, so named because the first two conserved cysteine residues are separated by three amino acids (8,9). Another unique characteristic of fractalkine is that it is the only chemokine that is expressed on the cell surface. By nucleotide sequence analysis, fractalkine consists of a chemokine head tethered to the cell surface by a mucin stalk, followed by a single transmembrane spanning domain and a short cytoplasmic tail (8, 9). In addition, fractalkine expression on endothelium is increased by proinflammatory cytokines interleukin-1 and tumor necrosis factor-␣. The receptor for fractalkine, CX 3 CR1 (V28), is expressed on T cells, monocytes, macrophages, and natural killer cells (10, 11). Fractalkine and CX 3 CR1 function as cell adhesion molecules under both st...

Regulated Shedding of Transmembrane Chemokines by the Disintegrin and Metalloproteinase 10 Facilitates Detachment of Adherent Leukocytes

“…When ECV-304 cells were transfected to express CX3CL1, the arrest of monocytic cells was clearly enhanced, as previously reported (7,46). This arrest was profoundly down-regulated by pretreatment of the ECV-304 cells with either PMA (200 ng/ml) or ionomycin (1 M) for 60 or 30 min, respectively (Fig.…”

Engineering a biospecific communication pathway between cells and electrodes

“…3d). This high level of glycosylation of the cutinase construct is consistent with the known O-glycosylation of the fractalkine stalk (15), which may also be the reason that the observed molecular mass of 62 kDa is slightly higher than the predicted value of 56.4 kDa, because a residual amount of glycosylation may remain after PNGase F treatment.…”

“…In either case, it is clear that the cell-surface cutinase can at least partly displace the adsorbed layer of protein and access the substrates on the monolayers. It is possible that this access is facilitated by the fractalkine stalk, which projects cutinase 26 nm away from the cell surface and might be able to project the enzyme more readily through an adsorbed protein layer than a construct lacking such a stalk (15). Alternatively, the signal enhancement we observed with fibronectin preadsorption may also be due to the increased local concentration of cutinase at the surface produced by rapid adhesion of the cell.…”

Enhanced Recruitment of CX3CR1+ T Cells by Mucosal Endothelial Cell–Derived Fractalkine in Inflammatory Bowel Disease

“…Transfection studies have revealed that the key function of the mucin portion of FKN is to mediate cell adhesion 8,9 . This portion extends the chemokine domain away from the endothelial cell surface and enables its presentation to leukocytes, thus directly supporting their adhesion to the endothelium 8, 9 .…”