CD14: Physical Properties and Identification of an Exposed Site That Is Protected by Lipopolysaccharide

McGinley, Michael D.; Narhi, Linda O.; Kelley, Michael J.; Davy, Elyse; Robinson, John R.; Rohde, Michael F.; Wright, Samuel D.; Lichenstein, Henri S.

doi:10.1074/jbc.270.10.5213

Cited by 58 publications

(54 citation statements)

References 13 publications

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“…This is confirmed by a recent X-ray structure of mouse sCD14 which depicts a α/β fold for the protein [16]. The spectrum of sCD14-EBD changes minimally upon addition of the endotoxin ligand ReLPS, suggesting there are no global conformational changes in the protein upon ligand binding [22]. Similar results were obtained upon binding of other ligands such as LTA and MDP (data not shown).…”

Section: Expression and Isotopic Labeling Of Scd14 In Pichia Pastorissupporting

confidence: 76%

Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14

Albright¹,

Chen²,

Holbrook³

et al. 2008

Biochemical and Biophysical Research Communications

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CD14 functions as a key pattern recognition receptor for a diverse array of gram-negative and grampositive cell-wall components in the host innate immune response by binding to pathogen-associated molecular patterns (PAMPs) at partially overlapping binding site(s). To determine the potential contribution of CD14 residues in this pattern recognition, we have examined using solution NMR spectroscopy the binding of three different endotoxin ligands, Lipopolysaccharide, lipoteichoic acid and a PGN-derived compound, muramyl dipeptide to a 15 N isotopically labeled 152-residue Nterminal fragment of sCD14 expressed in Pichia pastoris. Mapping of NMR spectral changes upon addition of ligands revealed that the pattern of residues affected by binding of each ligand is partially similar and partially different. This first direct structural observation of the ability of specific residue combinations of CD14 to differentially affect endotoxin binding may help explain the broad specificity of CD14 in ligand recognition and provide a structural basis for pattern recognition. Another interesting finding from the observed spectral changes is that the mode of binding may be dynamically modulated and could provide a mechanism for binding endotoxins with structural diversity through a common binding site. KeywordsNMR; human CD14 structure; CD14-endotoxin complex; endotoxin; pattern recognition; pichia pastoris expression; isotopic labeling; cell-based assay; lipopolysaccharide; ReLPS; LTA; BODIPY Clinical manifestation of sepsis typically involves a complex interaction between the host immune system and major bacterial cell wall constituents known as endotoxins. The cluster differentiation antigen, CD14, has been shown to be a key high-affinity cellular receptor for these bacterial endotoxins and plays an important role in endotoxin-induced activation of innate immune cells [1]. In humans, CD14 is constitutively expressed on cell surfaces of monocytes/ macrophages as a 55 kDa membrane-bound protein and also exists in a soluble form (sCD14), in serum and bodily fluids in concentrations of 2-6 μg/ml [2,3]. Recognition and binding of different microbial endotoxins to both mCD14 and sCD14 initiates a signaling cascade mediated by Toll-like receptors (TLRs) that promotes the synthesis and secretion of multiple host-derived inflammatory mediators [4], overactivation of which is ultimately responsible for the deleterious effects of sepsis. Understanding mechanism of endotoxin recognition is

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Section: Expression and Isotopic Labeling Of Scd14 In Pichia Pastorissupporting

confidence: 76%

Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14

Albright¹,

Chen²,

Holbrook³

et al. 2008

Biochemical and Biophysical Research Communications

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“…1) were independently necessary for LPS ligand binding. Subsequently, protease protection experiments, an additional deletion CD14 mutation protein, and mAb epitope mapping identified region 4 as required for E. coli LPS binding (14,15). Another study confirmed that region 3 is involved in E. coli LPS binding with the use of multiple alanine replacement mutations and monoclonal epitope mapping (16).…”

mentioning

confidence: 58%

“…A major portion of the 6C8 epitope is located in the hydrophilic region 4 (residues R53 to D59; see Table II). This region of CD14 has been identified as an E. coli LPS binding site based upon mutational analysis (15), mAb binding (15), and protease protection experiments (14). Residue D59 is probably a contact residue for Ab 6C8.…”

Section: Discussionmentioning

confidence: 99%

CD14 Employs Hydrophilic Regions to “Capture” Lipopolysaccharides

Cunningham

Shapiro

Seachord

et al. 2000

The Journal of Immunology

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CD14 participates in the host innate inflammatory response to bacterial LPS obtained from Escherichia coli and other Gram-negative bacteria. Evidence from several laboratories suggests that different regions of the amino-terminal portion of the molecule may be involved in LPS binding. In this report a series of single-residue serine replacement and charge reversal mutations were generated to further elucidate the mechanism by which this protein may bind a multitude of different LPS ligands. Single-residue CD14 mutation proteins were examined for their ability to bind LPS obtained from E. coli, Porphyromonas gingivalis, and Helicobacter pylori and facilitate the activation of E-selectin from human endothelial cells. In addition, the single-residue CD14 mutation proteins were employed to perform monoclonal epitope-mapping studies with three LPS-blocking Abs that bound tertiary epitopes. Evidence that several different hydrophilic regions of the amino-terminal region of CD14 are involved in LPS binding was obtained. Epitope-mapping studies revealed that these hydrophilic regions are located on one side of the protein surface. These studies suggest that CD14 employs a charged surface in a manor similar to the macrophage scavenger receptor to “capture” LPS ligands and “present” them to other components of the innate host defense system.

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“…Region 4 includes residues from the loop connecting ␣2 and ␣3 helices. This area is labile to proteolysis in the absence of bound LPS but becomes resistant when LPS is bound (31). Furthermore, the antibodies MEM18, CRIS-6, and 6C8 that block LPS binding bind to the same region (23)(24)(25).…”

Section: Structure Determination and Overall Structure Of Cd14 -mentioning

confidence: 99%

Crystal Structure of CD14 and Its Implications for Lipopolysaccharide Signaling

Kim

Lee

Jin

et al. 2005

Journal of Biological Chemistry

236

242

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Lipopolysaccharide, the endotoxin of Gram-negative bacteria, induces extensive immune responses that can lead to fatal septic shock syndrome. The core receptors recognizing lipopolysaccharide are CD14, TLR4, and MD-2. CD14 binds to lipopolysaccharide and presents it to the TLR4/MD-2 complex, which initiates intracellular signaling. In addition to lipopolysaccharide, CD14 is capable of recognizing a few other microbial and cellular products. Here, we present the first crystal structure of CD14 to 2.5 Å resolution. A large hydrophobic pocket was found on the NH 2 -terminal side of the horseshoelike structure. Previously identified regions involved in lipopolysaccharide binding map to the rim and bottom of the pocket indicating that the pocket is the main component of the lipopolysaccharide-binding site. Mutations that interfere with lipopolysaccharide signaling but not with lipopolysaccharide binding are also clustered in a separate area near the pocket. Ligand diversity of CD14 could be explained by the generous size of the pocket, the considerable flexibility of the rim of the pocket, and the multiplicity of grooves available for ligand binding.

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CD14: Physical Properties and Identification of an Exposed Site That Is Protected by Lipopolysaccharide

Cited by 58 publications

References 13 publications

Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14

Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14

CD14 Employs Hydrophilic Regions to “Capture” Lipopolysaccharides

Crystal Structure of CD14 and Its Implications for Lipopolysaccharide Signaling

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