Identification of a lipopolysaccharide binding domain in CD14 between amino acids 57 and 64.
CD14 is a 55-kDa glycoprotein which binds lipopolysaccharide (LPS) and enables LPS-dependent responses in a variety of cells. Recent limited proteolysis studies have implicated a region in CD14 between amino acids 57 and 64 as being involved in LPS interaction. To specifically assess the importance of this region with respect to LPS binding, we constructed a mutant sCD14 (sCD14 delta 57-64) lacking amino acids 57-64. sCD14 delta 57-64 was isolated from the serum-free conditioned medium of this cell line, and, in all assays, the purified protein failed to recognize LPS or enable LPS-dependent responses in cells. We also demonstrated that the region between amino acids 57 and 64 is required for binding of a neutralizing CD14 mAb, MEM-18. Native polyacrylamide gel electrophoresis assays were used to demonstrate that MEM-18 and LPS compete for the same binding site on CD14. These data strongly suggest that the region spanning amino acids 57-64 binds LPS and that formation of sCD14.LPS complex is required in order for sCD14-mediated responses to occur.
Juan
T S
TS
Amgen, Inc., Thousand Oaks, California 91320.
Hailman
E
E
Kelley
M J
MJ
Busse
L A
LA
Davy
E
E
Empig
C J
CJ
Narhi
L O
LO
Wright
S D
SD
Lichenstein
H S
HS
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D015214:Antigens, Differentiation, Myelomonocytic / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007668:Kidney; D007700:Kinetics; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014162:Transfection
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D017931:DNA Primers; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins