Identification of a domain in soluble CD14 essential for lipopolysaccharide (LPS) signaling but not LPS binding

J Biol Chem. 1995 Jul 21;270(29):17237-42. doi: 10.1074/jbc.270.29.17237.


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.

Publication types

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

MeSH terms

  • Acute-Phase Proteins*
  • Amino Acid Sequence
  • Antigens, CD / chemistry*
  • Antigens, CD / metabolism
  • Antigens, Differentiation, Myelomonocytic / chemistry*
  • Antigens, Differentiation, Myelomonocytic / metabolism
  • Base Sequence
  • Carrier Proteins / metabolism
  • Lipopolysaccharide Receptors
  • Lipopolysaccharides / metabolism*
  • Lipopolysaccharides / pharmacology
  • Membrane Glycoproteins*
  • Molecular Sequence Data
  • NF-kappa B / metabolism
  • Structure-Activity Relationship
  • Tumor Necrosis Factor-alpha / biosynthesis


  • Acute-Phase Proteins
  • Antigens, CD
  • Antigens, Differentiation, Myelomonocytic
  • Carrier Proteins
  • Lipopolysaccharide Receptors
  • Lipopolysaccharides
  • Membrane Glycoproteins
  • NF-kappa B
  • Tumor Necrosis Factor-alpha
  • lipopolysaccharide-binding protein