Soluble CD14 truncated at amino acid 152 binds lipopolysaccharide (LPS) and enables cellular response to LPS.
CD14 is a 55-kDa glycoprotein which binds lipopolysaccharide (LPS) and enables LPS-dependent responses in a variety of cells. In order to identify the domains in CD14 required for function, we deleted increasing amounts of CD14 from the C terminus. Truncated CD14 cDNA sequences were transfected into COS-7 cells and serum-free conditioned medium was analyzed for mutant CD14 expression and bioactivity. Mutant CD14s containing as few as 152 amino acids were found to have activity equivalent to full-length sCD14. To further characterize the mutant CD14, we constructed a stable Chinese hamster ovary cell line expressing sCD14(1-152) and purified the protein to homogeneity. sCD14(1-152) bound radioactive LPS, enabled U373 cells to synthesize interleukin 6 in response to LPS, and enabled human neutrophils to respond to smooth LPS. In all of these assays, the behavior of sCD14(1-152) was quantitatively similar to full-length sCD14. We also found that two neutralizing anti-CD14 antibodies (3C10 and MEM-18) bound and neutralized sCD14(1-152). We conclude from these experiments that the N-terminal 152 amino acids of CD14 are sufficient to bind LPS and confer essentially wild-type bioactivity in vitro.
Juan
T S
TS
Amgen, Inc., Thousand Oaks, California 91320.
Kelley
M J
MJ
Johnson
D A
DA
Busse
L A
LA
Hailman
E
E
Wright
S D
SD
Lichenstein
H S
HS
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015214:Antigens, Differentiation, Myelomonocytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002448:Cell Adhesion / Q000276:immunology; D002460:Cell Line; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000378:metabolism*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000166:cytology; D009838:Oligodeoxyribonucleotides
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D009838:Oligodeoxyribonucleotides