An amino-terminal fragment of human lipopolysaccharide-binding protein retains lipid A binding but not CD14-stimulatory activity.
LPS-binding protein (LBP) mediates the pro-inflammatory effects of bacterial LPS by enhancing LPS-induced cytokine production by monocytic cells. LBP binds specifically to LPS to generate a complex that interacts with the CD14 receptor on the surface of responsive cells. To identify the biologically active regions of the protein responsible for mediating these activities, we cloned and expressed human rLBP (456 amino acids) as well as a truncated form encoding amino acids 1-197 (rLBP25). Both forms of LBP bound to LPS with the same affinity, and similarly inhibited LPS activity in the Limulus amebocyte lysate assay. These results demonstrate that the LPS-binding domain of LBP resides entirely within the N-terminal 197 amino acids of the protein. rLBP and rLBP25 were compared for their ability to mediate CD14-dependent LPS effects on cells. rLBP was effective in mediating uptake of LPS and stimulation of TNF production by human monocytic THP-1 cells, whereas rLBP25 had no significant activity in these assays. Similarly, rLBP was able to mediate LPS-induced TNF production by human PBMC whereas rLBP25 was essentially inactive. These results suggest that the structural features of LBP required for mediating LPS effects via CD14 are probably located in the C-terminal region of the protein. Thus, the LPS-binding activity of LBP can be separated from the CD14-stimulatory activity, suggesting these activities are mediated by structural elements residing in different regions of the protein.
Theofan
G
G
XOMA Corporation, Santa Monica, CA 90404.
Horwitz
A H
AH
Williams
R E
RE
Liu
P S
PS
Chan
I
I
Birr
C
C
Carroll
S F
SF
Mészáros
K
K
Parent
J B
JB
Kasler
H
H
D000209:Acute-Phase Proteins; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000378:metabolism*; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008033:Limulus Test; D008050:Lipid A / Q000378:metabolism*; D018950:Lipopolysaccharide Receptors; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D002352:Carrier Proteins; D008050:Lipid A; D018950:Lipopolysaccharide Receptors; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D000099231:Lipopolysaccharide-Binding Protein