title: Identification of a lipopolysaccharide binding domain in CD14 between amino acids 57 and 64.
abstract: 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.
authors.affiliation: Amgen, Inc., Thousand Oaks, California 91320.
authors.forename: T S
authors.initials: TS
authors.lastname: Juan
authors.forename: E
authors.initials: E
authors.lastname: Hailman
authors.forename: M J
authors.initials: MJ
authors.lastname: Kelley
authors.forename: L A
authors.initials: LA
authors.lastname: Busse
authors.forename: E
authors.initials: E
authors.lastname: Davy
authors.forename: C J
authors.initials: CJ
authors.lastname: Empig
authors.forename: L O
authors.initials: LO
authors.lastname: Narhi
authors.forename: S D
authors.initials: SD
authors.lastname: Wright
authors.forename: H S
authors.initials: HS
authors.lastname: Lichenstein
mesh_terms: 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
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D017931:DNA Primers; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins