Mutation of amino acids 39-44 of human CD14 abrogates binding of lipopolysaccharide and Escherichia coli.
As a key receptor for lipopolysaccharide (LPS) on the surface of monocytes and macrophages, the CD14 molecule is primarily involved in non-specific host defense mechanisms against gram-negative bacteria. To delineate the structural basis of LPS binding, 23 mutants in the N-terminal 152 amino acids of human CD14 were generated and stably transfected into CHO cells. In each mutant, a block of five amino acids was substituted by alanine. Reactivity of the mutants with anti-CD14 mAbs, and their ability to interact with LPS and Escherichia coli were tested. 4 of 21 expressed CD14 mutants, ([Ala9-Ala13]CD14, [Ala39-Ala41, Ala43, Ala44]CD14, [Ala51-Ala55]CD14 and [Ala57, Ala59, Ala61-Ala63]CD14), are not recognized by anti-CD14 mAbs that interfere with the binding of LPS to human monocytes. However, only [Ala39-Ala41, Ala43, Ala44]CD14 is unable to react with fluorescein-isothiocyanate-labeled LPS or with FITC-labeled E. coli (055:B5). In addition, [Ala39-Ala4l, Ala43, Ala44]CD14 does not mediate LPS (E. coli 055:B5; 10 ng/ml)-induced translocation of nuclear factor kappaB in CHO-cell transfectants. The results indicate that the region between amino acids 39 and 44 forms an essential part of the LPS-binding site of human CD14.
Stelter
F
F
Institute of Immunology and Transfusion Medicine, Ernst-Moritz-Arndt-University, Greifswald, Germany. stelter@gryps1.rz.uni-greifswald.de
Bernheiden
M
M
Menzel
R
R
Jack
R S
RS
Witt
S
S
Fan
X
X
Pfister
M
M
Schütt
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D018604:Epitope Mapping; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000737:chemistry* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D016328:NF-kappa B