Ultrastructure and function of the fractalkine mucin domain in CX(3)C chemokine domain presentation.
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.
Fong
A M
AM
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Erickson
H P
HP
Zachariah
J P
JP
Poon
S
S
Schamberg
N J
NJ
Imai
T
T
Patel
D D
DD
D000469:Alkaline Phosphatase / Q000235:genetics / Q000648:ultrastructure; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C; D019743:Chemokines, CXC / Q000032:analysis / Q000378:metabolism*; D019040:E-Selectin / Q000235:genetics / Q000648:ultrastructure; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D008854:Microscopy, Electron; D009077:Mucins / Q000378:metabolism* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000648:ultrastructure; D014407:Tumor Cells, Cultured
C515538:CX3CL1 protein, human; D015815:Cell Adhesion Molecules; D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C; D019743:Chemokines, CXC; D019040:E-Selectin; D008565:Membrane Proteins; D009077:Mucins; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase