Identification of a lipopolysaccharide binding domain in CD14 between amino acids 57 and 64

J Biol Chem. 1995 Mar 10;270(10):5219-24. doi: 10.1074/jbc.270.10.5219.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antigens, CD / chemistry*
  • Antigens, CD / isolation & purification
  • Antigens, CD / metabolism*
  • Antigens, Differentiation, Myelomonocytic / chemistry*
  • Antigens, Differentiation, Myelomonocytic / isolation & purification
  • Antigens, Differentiation, Myelomonocytic / metabolism*
  • Base Sequence
  • Binding Sites
  • Cell Line
  • Chlorocebus aethiops
  • DNA Primers
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Kidney
  • Kinetics
  • Lipopolysaccharide Receptors
  • Lipopolysaccharides / metabolism*
  • Lipopolysaccharides / pharmacology
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Neutrophils / drug effects
  • Neutrophils / physiology*
  • Peptide Fragments / chemical synthesis
  • Peptide Fragments / pharmacology*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Deletion*
  • Structure-Activity Relationship
  • Transfection

Substances

  • Antigens, CD
  • Antigens, Differentiation, Myelomonocytic
  • DNA Primers
  • Lipopolysaccharide Receptors
  • Lipopolysaccharides
  • Peptide Fragments
  • Recombinant Proteins