Ultrastructure and function of the fractalkine mucin domain in CX3C chemokine domain presentation

Fractalkine (FKN), a CX3C chemokine/mucin hybrid molecule on endothelium, f unctions as an adhesion molecule to capture and induce firm adhesion of a s ubset of leukocytes in a selectin- and integrin-independent manner. We hypo thesized that the FKN mucin domain may be important for its function in adh esion, and tested the ability of secreted alkaline phosphatase (SEAP) fusio n proteins containing the entire extracellular region (FKN-SEAP), the chemo kine domain (CX3C-SEAP), or the mucin domain (mucin-SEAP) to support firm a dhesion under now. CX3C-SEAP induced suboptimal firm adhesion of resting pe ripheral blood mononuclear cells, compared with FKN-SEAP, and mucin-SEAP in duced no firm adhesion. CX3C-SEAP and FKN-SEAP bound to CX(3)CR1 with simil ar affinities. By electron microscopy, fractalkine was 29 nm in length with a long stalk (mucin domain), and a globular head (CX3C). To test the funct ion of the mucin domain, a chimeric protein replacing the mucin domain with a rod-like segment of E-selectin was constructed. This chimeric protein ga ve the same adhesion of peripheral blood mononuclear cells as intact FKN, b oth 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, extend ing the chemokine domain away from the endothelial cell surface to present it to flowing leukocytes.