Lipopolysaccharide (LPS) binding protein, truncated at Ile-197, binds LPS but does not transfer LPS to CD14.
Lipopolysaccharide (LPS) binding protein (LBP), a 58-60 kDa glycoprotein, binds to the lipid A region of LPS. The resulting LPS-LBP complex is recognized by both the membrane-bound (mCD14) and soluble forms of CD14 (sCD14), thereby enhancing the ability of LPS to activate myeloid, endothelial, and epithelial cells. To begin to characterize the structure-function relationships within LBP, we have created and expressed a truncated form of human LBP (herein called NH-LBP) comprising amino acid residues 1-197 of the parent molecule. Experiments were done to characterize the ability of NH-LBP to bind LPS and to promote LPS binding to CD14. We found that NH-LBP efficiently binds LPS but does not transfer the LPS to either mCD14 or sCD14. Additionally, NH-LBP inhibited LPS binding to LBP, inhibited the LBP-promoted binding of LPS to CD14, and inhibited the LBP-dependent activation of rabbit peritoneal exudate macrophages. The apparent dissociation constant for LPS-NH-LBP complexes is less than 1 x 10(-8) M which compares well with the dissociation constant for LPS-LBP complexes of approximately 1 x 10(-9) M. We conclude from these studies that the LPS binding site of LBP resides in the amino-terminal half of LBP and that the CD14 interaction site resides in the carboxyl-terminal half of LBP. These data suggest that appropriately modified fragments of LBP might provide novel reagents with high LPS binding affinity that could be useful in inhibiting LPS-dependent cellular activation in vivo.
Han
J
J
Department of Immunology, Scripps Research Institute, La Jolla, California 92037.
Mathison
J C
JC
Ulevitch
R J
RJ
Tobias
P S
PS
D000209:Acute-Phase Proteins; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism*; D001483:Base Sequence; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D017931:DNA Primers; D005434:Flow Cytometry; D006801:Humans; D007532:Isoleucine; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000378:metabolism; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D014162:Transfection; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D002352:Carrier Proteins; D017931:DNA Primers; D007532:Isoleucine; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D000099231:Lipopolysaccharide-Binding Protein