Isolation of a lipopolysaccharide (LPS)-resistant mutant, with defective LPS binding, of cultured macrophage-like cells

• Published in: The Journal of biological chemistry Vol. 265; no. 12; pp. 6606 - 6610
• Main Authors: HARA-KUGE, S, AMANO, F, NISHIJIMA, M, AKAMATSU, Y
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25-04-1990
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Carbohydrates; Cell Line; Drug Resistance - genetics; Fundamental and applied biological sciences. Psychology; Gram-Negative Bacteria; Kinetics; Lipopolysaccharide Receptors; Lipopolysaccharides - metabolism; Lipopolysaccharides - pharmacology; Macrophages - immunology; Molecular Structure; Mutation; Other biological molecules; Receptors, Immunologic - genetics; Receptors, Immunologic - metabolism; Radiolabelling; Molecular structure; Rodentia; Phospholipid; Binding site; Binding protein; Vertebrata; Biological fixation; Mammalia; Cell line; Mouse; Lipopolysaccharide; Scatchard plot; Mutation; Gram negative bacteria; Macrophage
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)39191-4

Lipopolysaccharide (LPS)-resistant mutants which did not respond to LPS were isolated from a macrophage-like mouse cell line,
J774.1. Unlike the parental J774.1 cells, these mutants grew even in LPS added medium as well as in normal growth medium without
any morphological changes. Assay of 125I-LPS binding to the cell monolayers revealed that one of these LPS-resistant mutants
(LR-9) was strikingly defective in LPS-binding activity. Scatchard plot showed that LR-9 cells lacked the high affinity binding
sites which were present in J774.1. The high affinity binding was inhibited by addition of excess unlabeled LPS, lipid A,
lipid IVA (tetraacyl-beta(1'-6)-linked D-glucosamine disaccharide-1,4'-bisphosphate), and lipid X (2,3-diacylglucosamine 1-phosphate)
and sensitive to proteinase K. LPS enhanced O2- generation and the release of arachidonic acid in J774.1 cells but not in
LR-9 cells. Other stimulants such as zymosan and 12-O-tetradecanoylphorbol 13-acetate, however, induced the release of arachidonic
acid in LR-9 cells as well as in J774.1 cells. LPS-photocross-linked assay allowed the identification of 65- and 55-kDa LPS-binding
proteins in the membrane fraction of J774.1 cells. Both of the bands were not detectable in that of LR-9 cells and disappeared
by competing with unlabeled LPS or lipid X. These results show that one or both of the two LPS-binding proteins might relate
to the specific membrane receptor for LPS.