The molecular basis for therapeutic concepts utilizing CD14.
The CD14 molecule is a key receptor on myeloid lineage cells involved in the recognition of lipopolysaccharide (LPS) and Gram-negative bacteria. The application of its soluble form, sCD14, has been shown to protect mice from lethality in LPS-induced shock. Therefore the protein or its derivatives may be considered as a possible therapeutic alternative for the treatment of patients suffering from Gram-negative septic shock. In this study we performed an alanine scan of amino acids 1 to 152 of human CD14. Twenty-three substitution mutants were generated and stably transfected into CHO-cells. In each mutant five amino acids were substituted by alanine. We analyzed (a) whether mutant proteins expressed on the surface of transfectants were recognized by a panel of anti-CD14 monoclonal antibodies (mAb's), (b) the ability of mCD14-mutants to bind LPS and E. coli in a serum- or LBP-dependent manner, and (c) the capacity of soluble mutants to mediate the LPS-induced IL 6 release of U 373 astrocytoma cells. Twenty-one CD14-mutants were expressed on the surface of transfectants and 18 were present as soluble forms in the culture supernatants. We demonstrated that only CD14(39-41,43-44)A completely lacked the ability to bind LPS and E. coli. In addition, a combined mutant CD14(9-13/57,59,61-63)A had very limited capacity to interact with LPS indicating that the LPS-binding site of human CD14 is a conformational epitope. Analysis of LPS-induced activation of CD14-negative U 373 cells revealed that the regions 9-13 and 91-101 are most important for sCD14-mediated signalling.
Stelter
F
F
Institute of Immunology and Transfusion Medicine, Ernst-Moritz-Arndt-University, Greifswald, Germany.
Bernheiden
M
M
Menzel
R
R
Witt
S
S
Jack
R S
RS
Grunwald
U
U
Fan
X
X
Schütt
C
C
D000209:Acute-Phase Proteins; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000737:chemistry / Q000502:physiology*; D008070:Lipopolysaccharides / Q000378:metabolism; D008562:Membrane Glycoproteins; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D013762:Tetrahydrofolate Dehydrogenase; D000099231:Lipopolysaccharide-Binding Protein