Identification of a domain in soluble CD14 essential for lipopolysaccharide (LPS) signaling but not LPS binding.
CD14 is a 55-kDa glycoprotein that binds lipopolysaccharide (LPS) and enables LPS-dependent responses in a variety of cells. Monoclonal antibodies of CD14 such as 3C10 and MEM-18 are known to neutralize biological activity of CD14. Recently, it has been demonstrated that MEM-18 recognizes the LPS-binding site of CD14, between amino acids 57 and 64. It has also been shown that 3C10 recognizes a distinct epitope from that of MEM-18, indicating that 3C10 may yet define another functional domain of CD14. In order to identify the epitope for 3C10, we constructed a series of alanine substitution mutants of soluble CD14 (sCD14). BIAcore analyses showed that regions between amino acids 7 and 10 and between amino acids 11 and 14 are required for 3C10 binding. To assess the effect of altering the 3C10 epitope in CD14, we generated a stable cell line expressing a mutant sCD14 containing alanine substitutions in the region between amino acids 7 and 10, sCD14(7-10)A, and purified this protein to homogeneity. sCD14(7-10)A has impaired ability to mediate LPS-dependent IL-6 up-regulation in U373 cells, integrin activation in neutrophils, and NF-kappa B activation in U373 cells. Purified sCD14(7-10)A was, however, capable of forming a stable complex with LPS in an LPS binding protein-facilitated and LPS binding protein-independent fashion. The ability of sCD14(7-10)A to bind LPS was also demonstrated in assays in which excess sCD14(7-10)A inhibited LPS-mediated tumor necrosis factor-alpha production in whole blood and adhesion of polymorphonuclear leukocytes to fibrinogen. These data strongly suggest that a region recognized by neutralizing monoclonal antibody 3C10 contains a domain required for cellular signaling but not for LPS binding.
Juan
T S
TS
Amgen, Inc., Thousand Oaks, California 91320, USA.
Hailman
E
E
Kelley
M J
MJ
Wright
S D
SD
Lichenstein
H S
HS
D000209:Acute-Phase Proteins; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry* / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000378:metabolism* / Q000494:pharmacology; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D002352:Carrier Proteins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D000099231:Lipopolysaccharide-Binding Protein