title: Identification of CD14 residues involved in specific lipopolysaccharide recognition.
abstract: CD14 is a key molecule responsible for the innate host inflammatory response to microbial infection. It is able to bind a wide variety of microbial ligands and facilitate the activation of both myeloid and nonmyeloid cells. However, its specific contribution to the innate recognition of bacteria is not known. Presently there is no information on the contribution of individual CD14 residues to Escherichia coli lipopolysaccharide (LPS) binding or on the molecular basis of the interaction between CD14 and LPS from other bacteria. LPS obtained from Porphyromonas gingivalis, a bacterium associated with chronic inflammatory disease, binds CD14 and activates myeloid cells but does not facilitate the activation of nonmyeloid cells. The transfer and binding of these two LPS species to soluble CD14 recombinant globulin proteins with single point mutations was examined. Functional activity of the mutant proteins was monitored by E-selectin expression on human umbilical cord endothelial cells. The analysis identified a charge reversal mutation in a single residue, E47, that demonstrated selective binding to E. coli LPS but not to P. gingivalis LPS. E-selectin activation assays indicated that proteins with mutations at position E47 maintained their structural integrity. Other mutations, including a charge reversal mutation of residue E58, did not significantly reduce the binding of either LPS ligand or the ability of the molecule to facilitate E-selectin activation. These data demonstrate that CD14 can selectively recognize different LPS ligands.
authors.affiliation: Inflammation Department, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Shapiro
authors.forename: M D
authors.initials: MD
authors.lastname: Cunningham
authors.forename: K
authors.initials: K
authors.lastname: Ratcliffe
authors.forename: C
authors.initials: C
authors.lastname: Seachord
authors.forename: J
authors.initials: J
authors.lastname: Blake
authors.forename: J
authors.initials: J
authors.lastname: Bajorath
authors.forename: A
authors.initials: A
authors.lastname: Aruffo
authors.forename: R P
authors.initials: RP
authors.lastname: Darveau
mesh_terms: D000595:Amino Acid Sequence; D002908:Chronic Disease; D019040:E-Selectin / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D006801:Humans; D007249:Inflammation / Q000209:etiology; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology; D017354:Point Mutation; D016966:Porphyromonas gingivalis; D011485:Protein Binding / Q000187:drug effects
chemical_list: D019040:E-Selectin; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D010446:Peptide Fragments