IDENTIFICATION OF A LIPOPOLYSACCHARIDE-BINDING DOMAIN IN CD14 BETWEEN AMINO-ACID-57 AND AMINO-ACID-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.