title: A region of human CD14 required for lipopolysaccharide binding.
abstract: CD14, a glycosylphosphatidylinositol-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, is a receptor for lipopolysaccharide (LPS). CD14 binding of LPS is enhanced by serum proteins, especially lipopolysaccharide binding protein. The serum-dependent binding of LPS to CD14 stimulates macrophages to make cytokines, which can cause septic shock in humans and animals. Here, we identify a region in human CD14 which is important in serum-dependent LPS binding and LPS-induced cellular activation. Four small regions (4-5 amino acids long) within the N-terminal 65 amino acids of CD14 were deleted singly or in combination. The deletion mutants were stably expressed in Chinese hamster ovary (CHO) cells. The mutants were characterized in three assays: reactivity with anti-CD14 monoclonal antibody, serum-dependent LPS binding, and LPS-induced activation of NF-kappa B. Some of the mutants selectively lost reactivity with the anti-CD14 monoclonal antibody that inhibited serum-dependent LPS binding and cellular activation. All of the mutants bound much less LPS than wild type CD14 in the presence of serum. None of the mutants bound more LPS than control CD14-CHO cells in the absence of serum. CD14-CHO cells respond to LPS by activation of NF-kappa B. All of the deletion mutants were less active LPS receptors than wild type CD14-CHO cells. The delta AVEVE mutant, the delta DDED and delta PQPD double mutant, and the delta DDED, delta PQPD, delta AVEVE, and delta DPRQY quadruple deletion mutants were essentially inactive LPS receptors in CHO cells. These studies suggest that the 65 N-terminal amino acids of CD14 are critical for serum-dependent binding of LPS to CD14 and subsequent signal transduction in CHO cells.
authors.affiliation: Department of Pathology, Department of Veterans Affairs Medical Center, San Diego, California 92161.
authors.forename: S
authors.initials: S
authors.lastname: Viriyakosol
authors.forename: T N
authors.initials: TN
authors.lastname: Kirkland
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D015214:Antigens, Differentiation, Myelomonocytic / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D001483:Base Sequence; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D017931:DNA Primers; D006801:Humans; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000378:metabolism*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D017931:DNA Primers; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides